Oligosaccharide conjugates and methods of use

ABSTRACT

There is provided a method of detecting in a sample the presence of an anti-M and/or anti-A and/or anti-C/Y antibody, the method comprising contacting the sample with a diagnostic conjugate provided according to the invention, comprising an oligosaccharide which comprises at least two units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising at least one -(1-3)- link between adjacent 4,6-dideoxy-4-acylamido-α-pyranose units, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH 2 OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a surface.

FIELD OF THE INVENTION

The invention relates to a novel diagnostic conjugate comprising anoligosaccharide, which can be used within assays for the reliablediagnosis of brucellosis. Furthermore, these assays can provide a DIVA(Differentiating Infected from Vaccinated Animals) test for vaccinationwith a novel vaccination conjugate also disclosed herein.

BACKGROUND

Brucellosis is a serious transboundary global zoonosis that is spread bylivestock and wildlife and is primarily due to infection with Brucellaabortus, B. melitensis or B. suis. Although the disease is serious inhumans, human to human transmission is rare and nearly all cases ofdisease are due to contact with infected animals or animal products(Franco et al. (2007) The Lancet Infectious Diseases 7, 775-786).Brucellosis (with the exception of canine brucellosis) is caused byinfection with smooth strains of Brucella, those that presentO-polysaccharide (OPS) as part of the smooth lipopolysaccharide (sLPS)on their outer membrane (Cardoso et al. (2006) Microbial Cell Factories5, 13).

The sLPS is a macromolecule comprising lipid A with a diaminoglucosebackbone attached to a set of core sugars which, in the case of smoothBrucella strains, is attached to the OPS which extends into theextracellular environment. This OPS is a recognised and establishedvirulence factor (Porte et al. (2003) Infection and Immunity 71,1481-1490) and induces a strong antibody response in the host againstwhich classical and contemporary serodiagnostic assays are directed(Nielsen & Yu (2010) Prilozi 31, 65-89). Unfortunately, infection withother Gram negative bacteria having OPS of similar structure, but whichmay be phylogenetically unrelated, may give rise to antibodies whichcross react in the classical and contemporary serodiagnostic assays(Corbel (1985) Vet. Bull. 55, 927-942). Such false positive serologicalreactions (FPSRs) become particularly problematic in regions of low tozero prevalence of brucellosis, whereby the positive predictive value ofthe serological assays becomes extremely small.

Attempts to improve the specificity of brucellosis serodiagnosis haverevolved mainly around efforts to identify and apply recombinant proteinantigens. Whilst many studies have been published extolling the virtuesof one antigen or another for the resolution of false positives, nonehave made it into assays that have progressed beyond the researchlaboratory into routine and effective use.

Antibodies raised owing to infection with Yersinia enterocolitica O:9cross react significantly with the OPS from Brucella smooth strains,owing to the considerable similarity in the structure (McGiven et al.(2008) Journal of Immunological Methods 337, 7-15) and are believed tocause many of the false positive serological reactions in diagnostictesting (Gerbier et al. (1997) Vet Res 28, 375-383). Likewise,antibodies raised against the OPS from smooth Brucella strains showconsiderable reaction against Y. enterocolitica O:9 OPS (Jungersen etal. (2006) Epidemiol Infect 134, 347-357).

The Brucella OPS is an unbranched variable length homopolymer formed by,on average, 50-100 units of the rare sugar4,6-dideoxy-4-formamido-D-mannopyranose (N-formyl perosamine) (Meikle etal. (1989) Infect Immun. 57, 2820-2828). The4,6-dideoxy-4-formamido-D-mannopyranose unit itself is only found innature within the OPS structure of Brucella organisms and of Y.enterocolitica O:9. In almost all Brucella organisms, neighbouring4,6-dideoxy-4-formamido-D-mannopyranose units are variably α-1,2 orα-1,3 linked. The structural data demonstrates that it is only the α-1,3linkage which differentiates Brucella and Y. enterocolitica O:9 OPS, asthe latter is an exclusively α-1,2 linked4,6-dideoxy-4-formamido-D-mannopyranose homopolymer (Caroff et al.(1984) Eur. J. Biochem. 139, 195-200). Between Brucella strains, theproportion of α-1,3 linkages varies between 2-20% (Meikle et al. (1989)Infect. Immun. 57, 2820-2828).

Even in Brucella strains with as much as 20% α-1,3 linkages, the OPSwill contain many contiguous α-1,2 linkages (Bundle et al. (1987)Biochemistry 26, 8717-8726). It is not known, however, if the sequenceof linkages is ordered and regular. There are three main biosynthesispathways for OPS, the Wzx/Wzy, ABC (Wzm/Wzt) and Synthase systems (Wanget al. (2010) In “Endotoxins: structure, function & recognition”;Springer Netherlands, pp. 123-152). Brucella utilises the ABC system(Gonzalez et al. (2008) PLoS ONE 3, e2760), as is more commonly found inorganisms that have homopolymeric OPS (including Y. enterocolitica O:9).In this pathway the saccharides are added to the growing OPS chainindividually rather than being constructed into repeating units and thenadded, as is the case in the Wzx/Wzy pathway where OPS is frequentlymade up of repeating blocks of three to five different monosaccharides.As such, it is quite possible that there is no regularity to thelocation of the α-1,3 links and they are distributed stochastically withregions of extended contiguous α-1,2 linkages even in strains with arelatively high proportion of α-1,3 linkages. The control and fidelityof the enzymes that synthesise the OPS have yet to be established.

The proportion of the two linkage types affects the shape of the OPS andthe different shapes are recognisable by monoclonal (Douglas et al.(1988) J Clin Microbiol 26, 1353-1356) and polyclonal antibodies such asthose used for serotyping Brucella into A (low number of α-1,3 linkages,typically 2%) and M (relatively high proportion of α-1,3 linkages,typically 20%) dominant strains or serotypes (Alton et al. (1994)Techniques for the Brucellosis Laboratory, pages 53-54; INRA Editions,ISBN-10: 2738000428).

The B. suis biovar 2 strain (Zaccheus et al. (2013) PLoS One 8, e53941)and B. inopinata BO2 (Wattam et al. (2012) mBio 3: 00246-12) do notappear to contain any α-1,3 linkages. B. inopinata BO2 is a highlyunusual and distantly related member of the Brucella genus as well asbeing extremely rare. Although it is a smooth strain, it is lacking mostof the genes required for the synthesis of4,6-dideoxy-4-formamido-D-mannopyranose OPS (Wattam et al (2012) mBio,3:5). Its OPS structure is of a different form which has not yet beenidentified.

The relative proportions and distribution of α-1,2 and α-1,3 linkageswithin the Brucella and Y. enterocolitica O:9 homopolymeric OPS createdistinct, but not necessarily completely described, antibody bindingepitopes. In Brucella, there are three different antigenic epitopeswhich can be found in the OPS for which there has been firm structuralevidence (Bundle et al. (1989) Infect. Immun. 57, 2829-2836) assummarised in Table 1:

TABLE 1 OPS epitopes Name of Number of Present in epitope perosaminesCharacteristics which OPS C/Y 3 to 4 N-formyl perosamines are All smoothBrucella exclusively joined by α1,2 strains and also linkages Y.enterocolitica O:9 A 5 or more N-formyl perosamines are Predominantlyjoined by α1,2 linkages within all A- dominant Brucella strains and alsoY. enterocolitica O:9 M 2-6 At least one α1,3 link Predominantly presentwith at least one within M-dominant adjacent α1,2 linkages; OPS Brucellalocation of α1,3 link strains but also, within epitope undefined to alesser extent, A-dominant strains. Not found in Y. enterocolitica O:9

Exclusively α-1,2 linked tri- and tetrasaccharide sequences are found inhigh abundance within the OPS from B. abortus, melitensis and suis aswell as in the OPS from Y. enterocolitica O:9. Such sequences are termed‘C/Y epitopes’ as they are common within all smooth strains ofeconomically significant Brucella and also to Y. enterocolitica O:9.Monoclonal antibodies that bind such sequences are termed anti-C/Y.

Longer sequences of more than four saccharides that are exclusivelyα-1,2 linked are more likely to be found in Brucella strains with lowerproportions of α-1,3 links. This also includes Y. enterocolitica O:9which contains only α-1,2 links. Such sequences are termed ‘A epitopes’and, of course, contain C/Y epitopes within them (C/Y epitopes beingα-1,2 linked perosamine chains up to 4 saccharides in length, asoutlined above). Monoclonal antibodies that bind such epitopes aretermed anti-A antibodies. Strains of Brucella with OPS containing lowproportions of α-1,3 links and, therefore, more abundant and longersequences of uninterrupted α-1,2 links, are termed “A dominant” strains(including most strains of B. abortus), even though the OPS may containa proportion of α-1,3 links at least once every 50 residues (Bundle etal. (1989) Infect. Immun. 57, 2829-2836). The OPS from A-dominantstrains of course contains C/Y epitopes, as well as A epitopes.

An anti-C/Y antibody will be expected to bind to both a C/Y epitope andto an A-epitope, since the shorter C/Y epitope structure forms part ofthe longer A-epitope structure.

Sequences of saccharides that contain a single α-1,3 linkage withlimited contiguous α-1,2 linkages are termed ‘M epitopes’. Monoclonalantibodies that bind to such sequences are termed anti-M antibodies.Strains of Brucella with a high proportion of α-1,3 links are termed Mdominant strains. Such strains comprise C/Y epitopes, but fewer Aepitopes, if any, since the series of α-1,2 linkages is “broken up” bythe presence of more frequent α-1,3 linkages.

The structure of the M epitope has not been defined and, in detailedterms, may vary according to the antibody. However, any M epitope mustbe sufficient in size to enable the binding of monoclonal antibodiesraised against antigens containing the α-1,3 linkage and be sufficientlylimited in α-1,2 linkages so as not to bind effectively to antibodiesraised against antigens (or parts of antigens) that are exclusivelyα-1,2 linked. Thus, the presence of the α-1,3 linkage is critical, as isa limitation on the number of α-1,2 linkages. The structure of theseantigens has been partially described previously (Bundle et al. (1989)Infect Immun 57, 2829-2836) although the allocation of Brucella strainsinto A and M types (and also mixed A and M) predates this considerably.This allocation is a fundamental part of the classical biotyping ofBrucella strains (Nielsen et al. (2009) “Bovine brucellosis” In: Manualof Diagnostic Tests & Vaccines for Terrestrial Animals 2009; OfficeInternational Des Epizooties, Paris, pg 10-19). Recent work suggeststhat the M dominant OPS contains a repeating structural determinant thatcontains one α-1,3 link for every three α-1,2 links. (Kubler-Kielb &Vinogradov (2013) Carbohydr. Res. 378, 144-147).

The typing of strains into A, M or mixed A and M (where there is aproportion of α-1,2 linkages of between 2-20% (Meikle et al. (1989)Infect Immun 57, 2820-2828)) is performed using sera from hyperimmunisedrabbits. The rabbits are inoculated with repeated doses of killedBrucella cells of either A or M dominant type until a high antibodytitre has been obtained. The polyclonal sera is then absorbed with theheterologous cell type (e.g., sera from rabbits hyperimmunised with Adominant strains are absorbed with M dominant cells) to removeantibodies that cross react. After careful selection and empiricaltesting, the process leaves a population of polyclonal (now termedmonospecific) antibodies that are more specific to the immunizing typeand can be used in agglutination assays to determine the A, M or mixed Aand M status of an untyped strain (Alton et al. (1994) Techniques forthe Brucellosis Laboratory, pages 53-54; INRA Editions, ISBN-10:2738000428).

Presumptive diagnosis of Brucellosis depends on detection of antibodiesto Brucella A, C/Y and M antigens and is confirmed by microbiologicalculture (Nielsen et al. (2009) “Bovine brucellosis” In: Manual ofDiagnostic Tests & Vaccines for Terrestrial Animals 2009; OfficeInternational Des Epizooties, Paris, pg 3-7). The A, C/Y and M antigenicdeterminants are expressed simultaneously on the O-antigenpolysaccharide domain of Brucella smooth lipopolysaccharides (s-LPS) andthis sLPS is used to detect antibodies present in sera of animals orhumans suspected of being infected. Unfortunately, Brucella is avirulent pathogen that must be grown under level 3 bio-containment. Thismakes the production of diagnostic O-antigens a demanding, specialisedand costly task, with significant health risks. Furthermore, since A,C/Y and M epitopes each may be expressed on a single Brucella sLPS andsince this antigen is resistant to common partial degradation methods,it has proved difficult to isolate pure A or M antigenic determinants.

In summary, the Brucella OPS is highly immunogenic and many antibodiesare raised against it in infected animals. This OPS contains a number ofoverlapping antibody epitopes, some of which are unique to Brucella andsome that are not. The existence of non-unique epitopes within theBrucella OPS compromises its use as a serodiagnostic antigen and is amajor factor in the occurrence of false positive serological results. Ithas previously been reported, by some workers (Alonso-Urmeneta et al.(1998) Clinical and Diagnostic Laboratory Immunology 5, 749-754), thatantibodies to common OPS epitopes dominate the humoral response in casesof animal brucellosis and that the epitopic structure of the LPS, be itA, M or mixed A and M dominance, is not relevant in serodiagnosis.

However, given the problems of cross-reactivity of antibodies raisedagainst different strains of Brucella, as well as other organisms, thereis a need to identify antigens and methods capable of discriminatingbetween antibodies raised against a Brucella bacterium and those raisedagainst other organisms.

SUMMARY OF INVENTION

According to a first aspect of the invention, there is provided a methodof detecting in a sample the presence of anti-M and/or anti-C/Y and/oranti-A antibodies, for example anti-O-polysaccharide antibodies such asanti-Brucella and/or anti-Y. enterocolitica O:9 antibodies, the methodcomprising contacting the sample with a diagnostic conjugate as definedbelow according to the second aspect (or third or fourth aspects) of theinvention. The method may comprise detecting binding of the conjugate toat least one antibody present in the sample. In an embodiment, thediagnostic conjugate for use in the first aspect of the invention may bea “universal antigen” for anti-M and anti-C/Y and anti-A antibodies, asdescribed below. The diagnostic conjugate comprises an oligosaccharidewhich comprises at least two 4,6-dideoxy-4-acylamido-α-pyranose unitsand comprises at least one -(1-3)- link between adjacent4,6-dideoxy-4-acylamido-α-pyranose units, the oligosaccharide beingcovalently linked to a non-saccharide carrier molecule or to a solidentity.

In an embodiment, there is provided a method of detecting in a samplethe presence of anti-M antibodies, for example anti-OPS antibodies suchas anti-Brucella antibodies, the method comprising contacting the samplewith a diagnostic conjugate as defined below according to the secondaspect (or third or fourth aspects) of the invention and detectingbinding of the conjugate to at least one anti-M antibody present in thesample. The diagnostic conjugate for use in this embodiment of theinvention is a “specific M-antigen”, as described below, capable ofdistinguishing between an anti-A (and/or anti-C/Y) antibody and ananti-M antibody. That is, the specific M-antigen diagnostic conjugatebinds preferentially (i.e., with greater specificity) to anti-Mantibodies as compared to anti-A (and/or anti-C/Y) antibodies. Thisembodiment of the method, therefore, may provide a method for detectingthe presence in a sample of anti-Brucella antibodies whilst avoidingdetection of antibodies against a non-Brucella organism. The inventorshave observed an unexpected specificity within the polyclonal antibodyresponse, in the vast majority of Brucella infected animals, to theα-1,3 linkage, the presence of which structurally differentiates moststrains of Brucella from Y. enterocolitica O:9 OPS. Given the very lownumber of α-1,3 links in the OPS of the majority of Brucella, it iscompletely surprising that the method of the invention is effective indetecting the presence of antibodies raised against almost all strainsof Brucella.

Detection of binding of the diagnostic conjugate to an antibody, asmentioned herein, may be by any known technique, for example, an ELISA,fluorescence polarisation assay (FPA), TR-FRET assay, lateral flow assayor bead-based agglutination assay, as described in more detail below.The invention is not limited to any of these assays and the skilledperson may readily contemplate alternative assays which might be used.

An anti-M antibody is one which is capable of binding to an M-epitopefrom a Brucella OPS or to an M-dominant antigen. An anti-A antibody isone which is capable of binding to an antigen comprising an A-epitope,or to an A-dominant antigen, as found in the Brucella OPS or the Y.enterocolitica O:9 OPS. An anti-C/Y antibody is one which is capable ofbinding to an antigen comprising a C/Y epitope, as found in the BrucellaOPS or the Y. enterocolitica O:9 OPS. This epitope may, in someoccurrences, be found within an A- or an M-dominant antigen. An anti-Mantibody may preferentially bind to an antigen comprising an M-epitopecompared to binding of the antibody to an antigen comprising an A or C/Yepitope (i.e., it will bind with greater specificity to an antigencomprising an M-epitope). Examples of such epitopes, antibodies andantigens are known in the art, as described above. Examples of anti-Mantibodies include BM40 (Greiser et al. (1987) Am. Inst. PasteurMicrobiol. 138, 549-560), BM28 or BM10 (Bundle et al. (1989) Infect.Immun. 57, 2829-2836). Examples of anti-A antibodies include YsT9.1 andYsT9.2 (Bundle et al. (1989) Infect. Immun. 57, 2829-2836).

According to a second aspect of the invention, there is provided adiagnostic conjugate comprising an oligosaccharide which comprises atleast two 4,6-dideoxy-4-acylamido-α-pyranose units and comprising atleast one -(1-3)- link between adjacent4,6-dideoxy-4-acylamido-α-pyranose units, the oligosaccharide beingcovalently linked to a non-saccharide carrier molecule or to a solidentity, such as a surface or a bead (encompassing, for example, anynon-liquid structure such as a gel or latex bead or surface).

The diagnostic conjugate described herein may prove useful in thedetection of antibodies to the A, C/Y and M epitopes described hereinand, in some embodiments, for distinguishing between antibodies to the Aor C/Y and M epitopes. Therefore, the diagnostic conjugate may be termeda “diagnostic antigen”.

The oligosaccharide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 4,6-dideoxy-4-acylamido-α-pyranose units.

A “specific M-antigen”, as mentioned throughout this specification, maybe provided where the diagnostic conjugate of the invention comprises anoligosaccharide comprising 2, 3, 4 or 54,6-dideoxy-4-acylamido-α-pyranose units. A “universal antigen” foranti-M and anti-A antibodies, as mentioned throughout thisspecification, may be provided where the diagnostic conjugate of theinvention comprises an oligosaccharide comprising 6 or more4,6-dideoxy-4-acylamido-α-pyranose units. These specific embodiments ofthe diagnostic conjugate are described further elsewhere herein.

The term “-(1-3)- link” as used throughout this specification indicatesan α-1,3 link (also known as an α-1→3 link) between adjacent4,6-dideoxy-4-acylamido-α-pyranose units in the oligosaccharide.Likewise, the term “-(1-2)- link” as used throughout this specificationindicates an α-1,2 link (also known as an α-1>2 link) between adjacent4,6-dideoxy-4-acylamido-α-pyranose units.

The at least one -(1-3)- link may be centrally positioned, as definedbelow. Where four or more 4,6-dideoxy-4-acylamido-α-pyranose units arepresent, the at least one -(1-3)- link may be internally positioned andoptionally also centrally positioned, as defined below.

The term “internally positioned -(1-3)- link” indicates that the -(1-3)-link is not the final link at either end of the molecule, that is, it ispositioned between 4,6-dideoxy-4-acylamido-α-pyranose units neither ofwhich forms a terminal unit of the oligosaccharide.

The term “centrally positioned -(1-3)- link” in the context of anoligosaccharide having an even number of4,6-dideoxy-4-acylamido-α-pyranose units indicates that the -(1-3)- linkis in the central position of the molecule with an equal number of4,6-dideoxy-4-acylamido-α-pyranose units to either side. In the contextof an oligosaccharide having an odd number of4,6-dideoxy-4-acylamido-α-pyranose units, the term “centrally positioned-(1-3)- link” indicates that the -(1-3)- link is positioned immediatelyto one side of the 4,6-dideoxy-4-acylamido-α-pyranose unit which is inthe middle of the molecule, i.e., the unit which has an equal number ofother units to either side of it. For example, in a trisaccharide thecentral unit is the second unit and the centrally positioned -(1-3)-link is the first link (between units one and two) or the second link(between units two and three); in a pentasaccharide the central unit isthe third unit and the centrally positioned -(1-3)- link is the secondlink (between units two and three) or the third link (between unitsthree and four).

In an embodiment, the oligosaccharide forming part of the diagnosticconjugate has no more than one -(1-3)- link, i.e., there is a single-(1-3)- link present in the oligosaccharide, with all other linksbetween 4,6-dideoxy-4-acylamido-α-pyranose units being a link which isnot an α-1,3 link, for example, which is an α-1,2 link.

Throughout this specification, the term “pyranose” indicates a sugar(for example, a pentose or hexose) comprising a pyran ring. In anyaspect or embodiment of the present invention, the C5 (carbon atposition 5 in the pyranose) in each 4,6-dideoxy-4-acylamido-α-pyranoseunit is linked to an R group, where R is independently selected from—CH₂OH, —H or an alkyl group having at least one C. The “independentselection” of the R group indicates that it may be different in each4,6-dideoxy-4-acylamido-α-pyranose contained in the oligosaccharide. Insome embodiments, the alkyl group may be a hydrocarbon having 1-5 Catoms, for example, 1, 2, 3, 4 or 5 C atoms.

In any aspect or embodiment of the invention, the alkyl group may be asaturated hydrocarbon which is branched or unbranched. The alkyl groupmay be a methyl, ethyl, propyl, butyl or pentyl group, for example. Inan embodiment, R is methyl.

In any aspect or embodiment of the invention, the acylamido in each4,6-dideoxy-4-acylamido-α-pyranose unit may be independently selectedfrom formamido, acetamido, propionamido or butyramido, i.e., eachacylamido present in the overall molecule may be any of formamido,acetamido, propionamido or butyramido, so that a mixture of groups maybe present in the oligosaccharide. In any embodiment,4,6-dideoxy-4-acylamido-α-pyranose may be4,6-dideoxy-4-formamido-α-D-mannopyranose.

In any embodiment of the oligosaccharide described herein, in any aspectof the invention, the reducing end of the oligosaccharide may be closedby an —OCH₃ group, formed by substitution by —CH₃ of the —H on the —OHmoiety which is linked to C1 (carbon at position 1) on the pyranosering. The reducing end may alternatively be “closed” with a-1-O—(CH₂)_(n)—COO—CH₃ group where n=3-9, which may enable linkage toprotein and/or non-protein molecules and/or to a solid entity. In afurther alternative, a non-perosamine sugar may be attached at anysuitable position to the oligosaccharide defined herein, optionally forthe purpose of linkage to other molecules. Other linking systems aredescribed below.

The oligosaccharide covalently linked to form part of the diagnosticconjugate may be a disaccharide having Formula I;

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaI).

In an embodiment, the disaccharide consists of Formula II:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (FormulaII).

The oligosaccharide may be a trisaccharide having Formula III or IV:

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaIII);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaIV).

The oligosaccharide may be a tetrasaccharide having Formula V, VI orVII:

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaV);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl  (FormulaVI);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaVII).

In an embodiment of Formula VII, the tetrasaccharide consists of FormulaVIII:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (Formula VIII)

In an embodiment, the oligosaccharide is a pentasaccharide of FormulaIX, X, XI or XII:

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (Formula IX);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranose  (Formula X);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (Formula XI);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (Formula XII).

By way of example, in both Formulae XI and XII above, a link which isnot an internally positioned link is shown in bold. It can be seen,therefore, that the -(1-3)- link in each of these molecules isinternally positioned. In addition, in both Formulae, the central4,6-dideoxy-4-acylamido-α-pyranose unit is underlined, showing that thatthe -(1-3)- link in these molecules is also centrally positioned,according to the above definition.

Any of the oligosaccharides of Formulae I-XII may be covalently linkedto a non-saccharide molecule or to a solid entity, to form a diagnosticconjugate which may be a specific M-antigen. The inventors found thatanti-Brucella OPS antibodies raised by infection with non-Brucellaorganisms did not bind effectively to a diagnostic conjugate comprisingthe disaccharide, trisaccharide, tetrasaccharide and pentasaccharidedescribed above. In light of this, the inventors' finding thatpolyclonal antibodies raised against A dominant as well as M dominantstrains of Brucella can bind to a diagnostic conjugate comprising thedisaccharide, trisaccharide, tetrasaccharide and pentasaccharidedescribed above was very surprising. This is surprising because Adominant Brucella strains have few M epitopes in the OPS. Thisinteraction is of sufficiently high specificity that antibodies thatcross react with the native OPS, but have not been raised againstBrucella, fail to bind these oligosaccharides effectively, if at all.Binding specificity is apparently greatest when no or few contiguous-(1-2)- links are present in the oligosaccharide.

The oligosaccharide may be a hexasaccharide having Formula XIII or XIV:

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaXIII);

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaXIV).

The -(1-3)- link is internally positioned in these molecules, as definedabove.

In an embodiment of the invention, the oligosaccharide may be anonasaccharide having Formula XV:

4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose  (FormulaXV).

Any of the oligosaccharides having Formulae XIII-XV are covalentlylinked to a non-saccharide molecule or to a solid entity, to form adiagnostic conjugate which may be a universal antigen, especially whenthe conjugate comprises the oligosaccharide of Formula XIV or XV.

The term “covalently linked” as used herein indicates that theoligosaccharide is joined to the non-saccharide molecule or solid entityvia a link or bridge which comprises at least one covalent bond.Therefore, the term “conjugate” or “diagnostic conjugate” as used hereinrefers to a structure comprising an oligosaccharide that has beencovalently linked or coupled to a “carrier” entity which is anon-oligosaccharide molecule such as (i) a protein or other molecule,for example one with a known biological activity such as the ability tofluoresce, or (ii) an inert amphiphilic polymer, or (iii) a solidmaterial entity such as a surface or a bead. In all three cases, thecoupling allows for various assay formats that detect the presence ofantibody in a sample, for example, ELISA, FPA, TR-FRET, lateral flowassay or bead-based agglutination assay, as outlined elsewhere herein.The oligosaccharide may be conjugated through the glycosidic oxygen atomor a sulfur atom. The covalent coupling may be directly to the protein,molecule, polymer or solid entity, or may be achieved via covalentcoupling to a linker as described below.

In the case of the conjugates described herein, the oligosaccharide iscovalently attached through a linker to a carrier entity such as aprotein carrier, polymer or solid entity such as a surface, usingchemical techniques providing for linkage of the oligosaccharide to thesaid carrier. The linker may form a direct bridge between theoligosaccharide and the carrier, or may be attached directly to theoligosaccharide and then be attached to the carrier via a couplingreagent, as outlined further below. In one embodiment, reactionchemistries are used that result in covalent linkages between the linkerand the carrier, as well as between the linker and the oligosaccharide.Such chemistries can involve direct attachment of theoligosaccharide-linker entity to the carrier, or attachment of theoligosaccharide-linker entity to a coupling reagent which itselfattaches to the carrier. For example, this may comprise the use ofcomplementary functional groups on hetero- or homo-bifunctionalcross-coupling reagents, described further below. Preferably, thecomplementary functional groups are selected relative to the functionalgroups available on the oligosaccharide and/or carrier for bonding, orwhich can be introduced onto the oligosaccharide and/or carrier forbonding.

Either of two approaches can be adopted for attachment of a linker tothe oligosaccharide. The first, as taught in (Lemieux et al. (1975) J.Am. Chem. Soc. 97, 4076-4083), assembles oligosaccharide on a linker.When the oligosaccharide-linker has been assembled, groups superfluousto further use, that were used to protect the oligosaccharide duringassembly, are removed from the completed oligosaccharide-linkerconstruct and the linker is activated for covalent attachment to protein(for example, to form an acyl azide group as shown in row 2 of Table 2).The second approach attaches the linker to the completed oligosaccharideafter synthesis (Ogawa et al. (1996) Carbohydr. Res. 293, 173-94) andthen removes protecting groups prior to conjugation with protein. In thefirst approach, the functionality used to establish the linkage toprotein, either latent or exposed, must survive all chemicaltransformation used to build and subsequently deprotect theoligosaccharide. In the second approach, the linker only has to survivethe deprotection step(s). In the work described herein, the inventorsemployed the first method and preserved the ester functionalitythroughout oligosaccharide synthesis and removal of protecting groups.

The term “linker” or “linking group” refers to the bridge structureproduced between the oligosaccharide and the carrier, after covalentbonding of a linking agent, homobifunctional cross coupling reagent, orheterobifunctional cross coupling reagent to the oligosaccharide and tothe carrier, as described below.

Suitable complementary functional groups for use in forming covalentlinkages are well known in the art. By way of example, reaction betweena carboxylic acid of either the linker or the protein carrier and aprimary or secondary amine of the protein carrier or the linker in thepresence of suitable, well-known activating agents results in formationof an amide bond; reaction between an amine group of either the linkeror the protein carrier and a sulfonyl halide of the protein carrier orthe linker results in formation of a sulfonamide bond covalently; andreaction between an alcohol or phenol group of either the linker or theprotein carrier and an alkyl or aryl halide of the carrier or the linkerresults in formation of an ether bond covalently linking the carrier tothe linker. Similarly, these complimentary reactions can occur betweenthe linker and the oligosaccharide to form a linkage between theoligosaccharide and the linker.

The following Table 2 illustrates numerous complementary reactive groupsand the resulting bonds formed by reactions between them.

TABLE 2 Complementary Reactive Groups and Resulting Linkages Firstreactive group Second reactive group Resulting linkage squarate amineAmide type acyl azide amine Amide carboxyl amine Amide ketone aminooxyoxime thiol bromo or iodoacetyl thioether carbonate amine Carbamateepoxide sulfhydryl β-hydroxythioether maleimide sulfhydryl Thioetherhydroxyl alkyl/aryl halide Ether amine epoxide β-hydroxyamine amineketone Imine amine ketone secondary amine sulfonyl halide amineSulfonamide epoxide alcohol β-hydroxyether hydroxyl isocyanate urethane

The term “linking agent” refers to a reagent that is used to couple twoother molecules or species together. Thus, linking agents includeheterobifunctional cross coupling reagents and homobifunctional crosscoupling reagents. In one embodiment, the linking agent comprises afunctional group selected from the “first reactive group” in Table 2. Inanother embodiment, the linking agent comprises a functional groupselected from the “second reactive group” in Table 2. For example, alinking agent can comprise a functional group selected from the “firstreactive group” in Table 2 while a mannopyranose derivative can comprisea functional group selected from the “second reactive group” in Table 2,or vice versa.

The term “heterobifunctional cross coupling reagents” refers to areagent that is used to couple two other molecules or species togetherby having at least two different functional groups built into onereagent. Such cross coupling reagents are well known in the art andinclude, for example, X-Q-X′, where each of X and X′ are preferablyindependently cross coupling groups selected, for example, from —OH,—CO₂H, epoxide, —SH, —N═C═S, and the like. Preferably, Q is a groupcovalently coupling X and X′ having from about 1 to about 20 atoms oralternatively, can be from about 1 to about 15 carbon atoms. Examples ofsuitable heterobifunctional cross coupling reagents includeN-ε-maleimidocaproic acid, m-maleimidobenzoyl-N-hydroxysuccinimideester, 3-(2-pyridyldithio)propionylhydrazide, N-succinimidyliodoacetate, NHS-PEG-maleimide and N-succinimidyl 3-(2-pyridyldithio)propionate. The heterobifunctional cross coupling reagents may also be alipid or lipid mimic, where the carbohydrate hapten may be covalentlylinked to the lipid or the lipid is co-administered as an immunologicaladjuvant.

The term “homobifunctional cross coupling reagents” refers to a reagentthat is used to couple two other molecules or species together by havingat least two of the same functional groups built into one reagent. Suchcross coupling reagents are well known in the art and include, forexample, X-Q-X, where X and Q are as defined above. Examples of suitablehomobifunctional cross coupling reagents include squarate derivatives,as well as entities derived from succinic anhydride, maleic anhydride,polyoxyalkylenes, adipic acid (CO₂H—C₆—CO₂H), and azelaic acid(CO₂H—C₉—CO₂H). 1,2-diaminoethane, a dicarboxylic acid chloride anddiethyl squarate are particular examples of such homobifunctional crosscoupling reagents. Homobifunctional cross coupling reagents may also bederived from lipids and lipid mimics.

A preferred embodiment employs the heterobifunctional5-methoxycarbonylpentanol as linker. The oligosaccharide is synthesisedby attaching one or more sugar units via an alpha linkage to thehydroxyl group of the linker. The linker is chosen such that its secondfunctional group remains unchanged throughout the chain extension of theoligosaccharide. Close variants of this linker type are alkanes bearinga terminal alcohol and a terminal ester group composed of 4 to 10 carbonatoms, or di or triethylene glycols bearing a terminal alcohol and aterminal ester group.

After removal of protecting group from the sugar residues andintroduction of crucial formamido functionalities, the linker ester isconverted to an amide which is conjugated to protein or polymer withprimary amino group using a homobifunctional coupling reagent, di-alkylsquarate.

Other methods are known to practitioners of the art. For example (astaught by Lemieux) the linker ester may be converted to an acylhydrazide and, by reaction with nitrous acid or dinitrogen tetroxide, ahighly reactive acyl azide species is produced. Without isolation, thisrapidly conjugates with protein or polymer primary amino groups. Lessuseful are methods whereby the linker ester under goes reaction to yieldan aldehyde which may be conjugated by reductive amination. Methods ofthis type are less effective, because the chemistry to convert the estergroup to appropriate functionalities may be incompatible with functionalgroups present in the oligosaccharide.

By way of non-limiting example, as in the work exemplified herein, theoligosaccharide may be linked to Bovine Serum Albumin (BSA) (orco-povidone) by being synthesised with a 5-methoxycarbonylpentanollinker molecule, the other end of which is reacted with a dialkylsquarate coupling reagent to provide a squarate half ester, which formsamide bonds to amino groups present in the BSA protein (or co-povidone).

In the conjugate according to the invention, the oligosaccharide iscovalently attached as described above to a carrier, for example, anon-oligosaccharide entity such as a protein, for example, Bovine SerumAlbumin (BSA), to a non-protein carrier molecule comprising hydrophobicelements, or to a fluorophore to enable detection in, for example, aTR-FRET or FPA system. The oligosaccharide may be covalently attached toa carrier which is a solid entity such as a surface and/or a membraneand/or a bead, by way of non-limiting example. A “solid” beadencompasses non-liquid structures such as gel beads or latex beads.Therefore, the diagnostic conjugate may be in the form of a surfacehaving at least one oligosaccharide as described herein attached theretovia a linking system which includes a covalent attachment to theoligosaccharide. Attachment may be, for example, via passive absorptionmediated by a protein carrier, or a non-protein carrier moleculecomprising hydrophobic elements, covalently attached to theoligosaccharide. The passive absorption being due to, for example,hydrophobic and ionic interactions with a surface such as polystyrene,polyvinyl chloride, latex, glass, nitrocellulose, polyvinylidenedifluoride. The protein carrier may be, for example, BSA. The attachmentmay alternatively be via hydrazone conjugation, which includes providingaldehyde groups on the oligosaccharide by periodate oxidation. Wherehydrazone conjugation is used, the surface may be a Carbo-BIND™ ELISAplate. Other functional groups available on the solid entity surface mayalso be utilised, such as maleimide (binds to sulfhydryls), amine(numerous binding options available through use of a linker, as outlinedin Table 2), aldehydes (bind to amines), or carboxyl (bind to amines).

The diagnostic conjugate described herein may be a synthetic conjugate,for example, the oligosaccharide and conjugate being synthesised bymethods such as those outlined below.

In the method according to the invention, the sample may be a biologicalsample obtained from an animal, for example, an animal which is or hasbeen, or is suspected of being or having been, infected with a Brucellabacterium. The animal may be a ruminant, camelid or suid animal such asa bovine or swine animal, for example, a cow, pig, sheep or goat, or maybe a human being. The biological sample may be a blood, plasma, serum,tissue, saliva or milk sample. In particular, a biological sample is nota laboratory sample comprising only antibodies and/or oligosaccharides(plus laboratory reagents), but is a complex sample also comprising manyother components including other antibodies, unrelated to the method tobe conducted. The presence of anti-M antibodies in a sample from ananimal indicates that the animal is, or has previously been, infectedwith a smooth strain Brucella bacterium so as to elicit an immuneresponse and raising of antibodies. The Brucella may be any smoothstrain (those that present OPS on their surface) which is not B. suisbiovar 2 or B. inopinata BO2.

The method may take the form of an ELISA assay, for example an indirectELISA or a competitive ELISA, the design of which is within the routineability of the skilled person. For example, in an indirect ELISA, theoligosaccharide described herein is immobilised on an ELISA plate toform a diagnostic conjugate according to the invention, for example viahydrazone binding to a Carbo-BIND™ plate as described herein, or to anon-functionalised ELISA plate via the use of a conjugated carriermolecule such as BSA, capable of passive absorption to the plate. Thebiological sample to be tested is then added to the plate and incubatedfor a period of time, after which the plate is washed. A detectionconjugate (such as HRP-conjugated Protein-G) is added and the plateincubated, washed and subsequently developed by a method appropriate tothe detection conjugate being used (in the case of HRP, ABTS may besuitable, as described below). This allows determination of the level ofbinding, if any, of antibodies present in the biological sample to theoligosaccharide present on the plate. Specific examples are described indetail below.

In a competitive ELISA, the oligosaccharide may be immobilised on anELISA plate to form a diagnostic conjugate according to the invention,for example, via hydrazone binding to a Carbo-BIND™ plate as describedherein, or to a non-functionalised ELISA plate via the use of aconjugated carrier molecule such as BSA, capable of passive absorptionto the plate. The biological sample to be tested is then added to theplate in conjunction with an anti-M monoclonal antibody (such as theBM40 antibody) and incubated for a period of time, after which the plateis washed. Binding of the anti-M monoclonal antibody is then detected bythe addition to the plate of a suitable enzyme conjugate thatspecifically binds to the anti-M monoclonal antibody and a furtherincubation, after which time the plate is washed. (If the anti-Mantibody has already been enzyme labelled, for example with HRP, theadditional conjugate and incubation step would not be required.) Theplate is subsequently developed by a method appropriate to the conjugatebeing used (in the case of HRP, ABTS may be suitable, as describedbelow). This allows determination of the level of binding of the anti-Mmonoclonal antibody. Any anti-M antibodies in the sample will competewith the monoclonal anti-M antibody for binding to the immobilisedantigen, resulting in a reduction of assay signal. This is used as ameasure of the presence of anti-M antibodies in the biological sample.

Other ELISA variants, such as a blocking ELISA (Rhodes et al. (1989) J.Vet. Diagn. Invest. 1, 324-328), are well known to the skilled personand may be utilised without application of inventive skill.

The method according to the invention may comprise use of TR-FRETmethods, such as are described, for example, in WO2009/118570 andWO2011/030168. In this context, the oligosaccharides may be conjugated,directly or indirectly, to a TR-FRET label such as a lanthanide chelate(donor fluorophore) or fluorescein (acceptor fluorophore) as describedin those patent publications.

As mentioned above, the oligosaccharides may also be conjugated,directly or indirectly, to fluorophores that will enable the detectionof antigen-antibody binding by fluorescence polarisation (Nasir andJolley (1999) Comb. Chem. High Throughput Screen., 2, 177-190) asdescribed, for example, in U.S. Pat. No. 5,976,820. This forms the basisof a fluorescence polarisation assay (FPA) as referred to elsewhereherein.

By way of non-limiting example, other assay formats which may beutilised in the invention include a lateral flow assay, in which antigenis absorbed to a membrane along which a serum (comprising serumantibodies) may be caused to flow. The serum may be mixed withanti-species antibodies, labelled with colloidal gold or latex beads(Abdoel et al. (2008) Vet. Microbiol. 130, 312-319). A furtheralternative is a bead based agglutination assay, for example in which anantigen-BSA conjugate is passively coated to a latex bead. The bead isthen added to a serum sample and the occurrence or absence ofagglutination observed (indicating antibody binding to antigen on thebead) (Abdoel & Smiths (2007) Diagn. Microbiol. Infect. Dis. 57,123-128).

The method may be a diagnostic method of determining that an animal isor has been infected with a Brucella organism, the method comprisingcontacting a biological sample previously obtained from the animal witha diagnostic conjugate according to the second aspect (and/or thirdand/or fourth aspects, described below) of the invention. The animal maybe a human being or a cow, pig, sheep or goat. The biological sample maybe a blood, plasma, serum, tissue, saliva or milk sample.

Also provided is the diagnostic conjugate according to the second aspectof the invention for use in a diagnostic method of determining that ananimal is or has been infected with a Brucella organism, as outlinedabove.

The methods described herein utilise the diagnostic conjugate alsodescribed herein and provide, for the first time, a method for detectionof anti-A and/or anti-C/Y and/or anti-M antibodies, for example anti-OPSantibodies such as anti-Brucella antibodies, using a synthetic antigen.As mentioned above, since Brucella must be grown under level 3bio-containment, production of diagnostic O-antigens is a demanding,specialised and costly task. Provision of a synthetic antigen is,therefore, a significant advantage. The data presented herein show thatchemical syntheses have successfully provided a universal antigen todetect Brucella antibodies that arise during infection by all Brucellastrains producing a sLPS (as well as other bacteria with an antigenhaving “A” epitope characteristics). This provides an extremely valuableand convenient antigen for presumptive diagnosis and one that can bedeployed in virtually any assay format, including those that do notrequire sophisticated equipment, unavailable in remote locations(Martinez et al. (2007) Proc. Natl. Acad. Sci. 105, 19606-19611).

Furthermore, the provision in some embodiments of the present inventionof a specific M-antigen, capable of preferentially binding to anti-Mantibodies, provides for the first time a method enabling detection ofantibodies against a Brucella bacterium whilst avoiding detection ofantibodies against a non-Brucella organism (such as a Gram-negativeorganism in possession of an OPS of highly similar structure to that ofBrucella, such as Y. enterocolitica O:9). Therefore, in the method ofthe invention, the proportion of such false positive results due todetection of infection by such a non-Brucella bacterium is significantlyreduced compared to a serodiagnostic assay method not according to theinvention, e.g., as described in Nielsen et al. (“Bovine brucellosis”In: Manual of Diagnostic Tests & Vaccines for Terrestrial Animals 2009;Office International Des Epizooties, Paris, pg 10-19). In this context,the Brucella bacterium may be a B. abortus, B. melitensis or B. suis(with the exception of strains of B. suis biovar 2) bacterium. Thepresence of antibodies to B. suis biovar 2 or B. inopinata BO2 may notbe detected using this embodiment of the method, as outlined above. Theutility of the diagnostic conjugates disclosed herein, which are aspecific M-antigen, for detection of almost all smooth strains ofBrucella, both A-dominant and M-dominant strains, is a completelyunexpected result, due to the low occurrence of the M-epitope inA-dominant strains.

In any case, the application of a discrete, isolated, M epitope (i.e., aspecific M-antigen as described herein) for use in diagnosis has notpreviously been proposed. This is because the epitope has not previouslybeen clearly defined, has been reported as having no diagnosticsignificance and is of variable occurrence in the OPS from different andsignificant Brucella strains. This is due to its dependency on thefrequency of the α-1,3 linkage, which in most A dominant strains is aslow as 2% (apart from in B. suis biovar 2). A diagnostic assay that canonly detect sera derived from infection with M dominant strains is oflimited application, since infection with A dominant strains is alsopossible. As mentioned, it is completely unexpected that a specificM-antigen as described herein would be useful for detection ofA-dominant, as well as M-dominant, strains of Brucella.

The method according to the invention may be a method for determiningwhether an animal has been exposed to and/or infected with a Brucellaorganism, the method comprising a first step of contacting a firstsample obtained from the animal with a diagnostic conjugate which is auniversal antigen and detecting binding of the conjugate to at least oneantibody present in the sample, the method optionally further comprisinga second step of contacting a second sample obtained from the animalwith a diagnostic conjugate which is a specific M-antigen and detectingbinding of the conjugate with at least one anti-M antibody present inthe sample. The first and second samples may be the same sample. Thesecond step may be carried out only if the first step provides apositive indication that anti-A and/or anti-C/Y and/or anti-M antibodies(for example, anti-OPS antibodies such as anti-Brucella antibodies) arepresent in the sample. This is because a negative outcome to this firstcontacting step may be taken as an indication that the animal has notbeen or is not infected with or exposed to a Brucella organism. Themethod may encompass an initial step of obtaining the sample(s) from theanimal.

Detection of binding of the diagnostic conjugate to an antibody, asmentioned herein, may be by any known technique such as an ELISA,fluorescence polarisation assay (FPA), TR-FRET assay, lateral flow assayor bead-based agglutination assay, as described in more detail above.

The universal antigen may comprise an oligosaccharide selected fromFormulae XIII-XV. The specific M-antigen may comprise an oligosaccharideselected from Formulae I-XII. The method as described here may provide,therefore, a method for reducing the occurrence of false positiveserological reactions (FPSRs) when conducting a method for determiningwhether an animal is infected with, or has been exposed to, a Brucellaorganism. “Reducing the occurrence” may be an indication that a lowerproportion of FPSRs (as determined using conventional, prior art,serological methods) is determined as positive by the method of theinvention, whilst effective determination of true positives ismaintained. Alternatively or additionally, “reducing the occurrence” maybe an indication that a greater proportion of animals determined by themethod of the invention to be infected with a Brucella organism are infact so infected, as compared to the proportion of animals determined tobe infected using prior art methods. Confirmation of infection with aBrucella organism can be confirmed, for example, by microbiologicalculture (Nielsen et al. (“Bovine brucellosis” In: Manual of DiagnosticTests & Vaccines for Terrestrial Animals 2009; Office International DesEpizooties, Paris, pg 3-7).

The method according to the invention may be a method fordifferentiating animals infected with Brucella from animals vaccinatedwith a vaccine conjugate according to the sixth aspect of the invention,as outlined below. Such a vaccine conjugate comprises an oligosaccharidehaving at least two 4,6-dideoxy-4-acylamido-α-pyranose units, each unitbeing joined to an adjacent unit by a -(1-2)- link, the oligosaccharidebeing covalently linked to a vaccine carrier molecule. The use of themethod, comprising use of the diagnostic conjugate according to theinvention, enables the development of useful synthetic vaccines againstBrucella, since the method provides a DIVA (Differentiating Infectedfrom Vaccinated Animals) test to distinguish infected animals fromanimals vaccinated with a vaccine comprising exclusively -(1-2)- linked4,6-dideoxy-4-acylamido-α-pyranose units.

According to a third aspect of the invention there is provided adiagnostic conjugate comprising an oligosaccharide comprising at leastsix 4,6-dideoxy-4-acylamido-α-pyranose units and comprising overlappingtetrasaccharides of Formula VII, such that the third and fourth4,6-dideoxy-4-acylamido-α-pyranose units in one tetrasaccharide form thefirst and second 4,6-dideoxy-4-acylamido-α-pyranose units in the nexttetrasaccharide, to form an oligosaccharide in which all links betweenmultiple 4,6-dideoxy-4-acylamido-α-pyranose units are alternating-(1,2)- and -(1,3)- links, the oligosaccharide being covalently linkedto a non-saccharide molecule or to a solid entity.

The oligosaccharide may be 6-100 4,6-dideoxy-4-acylamido-α-pyranoseunits in length, for example about 6, 8, 10, 12, 24, 36, 48, 60, 72, 84or about 96 units in length. Whatever the overall length, the pattern of4,6-dideoxy-4-acylamido-α-pyranose units and links is as follows, where“S” indicates a single 4,6-dideoxy-4-acylamido-α-pyranose unit, “2”indicates a -(1,2)- link and “3” indicates a -(1,3)- link:

That is, there are never consecutive links which are of the same type.Overlapping tetrasaccharides are underlined in the sequence above.4,6-dideoxy-4-acylamido-α-pyranose unit numbers for the firsttetrasaccharide in the chain are shown above the sequence in italics,with unit numbers for the second tetrasaccharide in the chain shownabove the sequence in bold. This shows how the tetrasaccharides overlapsuch that the third and fourth units in one tetrasaccharide form thefirst and second units in the next tetrasaccharide.

Therefore, the oligosaccharide comprises at least one“S-2-S-3-S-2-S-3-S-2-S” subunit wherein each subunit is linearly linkedto another subunit by an α-1,3 link.

According to a fourth aspect of the invention there is provided adiagnostic conjugate comprising an oligosaccharide comprising at leastseven 4,6-dideoxy-4-acylamido-α-pyranose units and comprisingoverlapping tetrasaccharides of Formula VII, such that the fourth4,6-dideoxy-4-acylamido-α-pyranose unit in one tetrasaccharide forms thefirst 4,6-dideoxy-4-acylamido-α-pyranose unit in the nexttetrasaccharide, the oligosaccharide being covalently linked to anon-saccharide molecule or to a solid entity.

The oligosaccharide may be 7-100 4,6-dideoxy-4-acylamido-α-pyranoseunits in length, for example about 7, 10, 13, 16, 19, 21, 27, 33, 39,45, 51, 57, 63, 69, 75, 81, 87, 93, 99 or about 105 units in length.Whatever the overall length, the pattern of4,6-dideoxy-4-acylamido-α-pyranose units and links is as follows, where“S” indicates a single 4,6-dideoxy-4-acylamido-α-pyranose unit, “2”indicates a -(1,2)- link and “3” indicates a -(1,3)- link:

That is, there are never more than two consecutive α-1,2 links.Overlapping tetrasaccharides are underlined in the sequence above.4,6-dideoxy-4-acylamido-α-pyranose unit numbers for the firsttetrasaccharide in the chain are shown above the sequence in italics,with unit numbers for the second tetrasaccharide in the chain shownabove the sequence in bold. This shows how the tetrasaccharides overlapsuch that the fourth unit in one tetrasaccharide forms the first unit inthe next tetrasaccharide.

Therefore, the oligosaccharide comprises at least one“S-2-S-3-S-2-S-2-S-3-S” subunit wherein each subunit is linearly linkedto another subunit by an α-1,2 link.

The diagnostic conjugate according to the third or fourth aspects of theinvention each provide a specific M-antigen, capable of preferentiallybinding to an anti-M antibody as compared to the level of binding to ananti-C/Y or anti-A antibody.

A fifth aspect of the invention provides a kit for carrying out a methodaccording to the invention, comprising a diagnostic conjugate accordingto the second and/or third and/or fourth aspects of the invention. Thekit may also comprise means for obtaining and/or containing a biologicalsample from an animal. This kit may comprise, for example, laboratoryreagents useful for conducting an antibody-antigen binding detectionassay, such as an ELISA. The kit may comprise packaging materials and/ormaterials providing instructions for use of the kit.

The diagnostic conjugate contained in the kit may be in the form of asolid entity having attached thereto at least one oligosaccharide whichcomprises at least two 4,6-dideoxy-4-acylamido-α-pyranose units andcomprising at least one -(1-3)- link between adjacent4,6-dideoxy-4-acylamido-α-pyranose units. The solid entity may haveattached thereto an oligosaccharide according to any of FormulaeXIII-XV, thus providing a universal antigen, or an oligosaccharideaccording to any of Formulae I-XII, thus providing a specific M-antigen.The solid entity may be, for example, a surface such as an ELISA plate.The kit may comprise a diagnostic conjugate which is a universalantigen, or may comprise a diagnostic conjugate which is a specificM-antigen, and/or may comprise both a diagnostic conjugate which is auniversal antigen and a diagnostic conjugate which is a specificM-antigen. A kit comprising both types of diagnostic conjugate isadvantageously useable initially to detect in an animal infection by orexposure to an organism expressing an OPS (for example, a Brucellaorganism) and subsequently to confirm that the animal is infected with,or has been exposed to, a Brucella organism, as opposed to an organismsuch as Yersinia enterocolitica O:9.

According to a sixth aspect of the invention, there is provided avaccine conjugate comprising an oligosaccharide having at least two4,6-dideoxy-4-acylamido-α-pyranose units and comprising a -(1-2)- linkbetween adjacent units, the oligosaccharide being covalently linked to avaccine carrier molecule. The oligosaccharide within the vaccineconjugate comprises only -(1-2)- links. The linkage to the carriermolecule is achieved in accordance with the methods outlined above inrelation to the diagnostic conjugate of the invention. A vaccine carriermolecule may be, for example, a protein or peptide which may be anyknown in the art to be useful as a conjugate to an antigenic molecule toform a vaccine. For example, the vaccine carrier molecule may be tetanustoxoid (Verez-Bencomo et al. (2004) Science 305, 522-525), CRIM 197(Mawas et al. (2002) Infect. Immun. 70, 5107-5114) or other highlyimmunogenic proteins (Svenson & Lindberg (1981) Infect. Immun. 32,490-496). The vaccine carrier molecule may also be an immunogenicparticle such as a liposome or inactive viral particle wherein theoligosaccharide is incorporated at the surface of the particle.

A seventh aspect of the invention provides a vaccine compositioncomprising a vaccine conjugate according to the sixth aspect of theinvention. The vaccine composition may further comprise excipientsand/or diluents appropriate for the means by which the composition is tobe administered to a subject in need of vaccination against infection byBrucella. Selection of appropriate components is within the routinecapability of the skilled person without the application of inventiveactivity.

For example, the vaccine composition of the invention may convenientlybe formulated using a pharmaceutically acceptable excipient or diluent,such as, for example, an aqueous solvent, non-aqueous solvent, non-toxicexcipient, such as a salt, preservative, buffer and the like. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil and injectable organic esters such as ethyloleate. Aqueoussolvents include water, alcoholic/aqueous solutions, saline solutions,parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.Preservatives include antimicrobials, anti-oxidants, chelating agentsand inert gases. The pH and exact concentration of the variouscomponents the vaccine composition are adjusted according to routineskills.

In certain situations, it may also be desirable to formulate the vaccinecomposition to comprise an adjuvant to enhance the immune response. Suchadjuvants include all acceptable immunostimulatory compounds such as,for example, a cytokine, toxin, or synthetic composition. Commonly usedadjuvants include aluminium hydroxide, aluminium phosphate, calciumphosphate, Freund's adjuvants and Quil-A saponin. In addition toadjuvants, it may be desirable to co-administer biologic responsemodifiers (BRM) with the vaccine conjugate to down regulate suppressor Tcell activity.

Possible vehicles for administration of the vaccine composition includeliposomes. Liposomes are microscopic vesicles that consist of one ormore lipid bilayers surrounding aqueous compartments. Liposomes aresimilar in composition to cellular membranes and, as a result, liposomesgenerally can be administered safely and are biodegradable. Techniquesfor preparation of liposomes and the formulation (e.g., encapsulation)of various molecules with liposomes are well known.

Depending on the method of preparation, liposomes may be unilamellar ormultilamellar and can vary in size with diameters ranging from 0.02 μmto greater than 10 μm. Liposomes can also adsorb to virtually any typeof cell and then release the encapsulated agent. Alternatively, theliposome fuses with the target cell, whereby the contents of theliposome empty into the target cell. Alternatively, an absorbed liposomemay be endocytosed by cells that are phagocytic. Endocytosis is followedby intralysosomal degradation of liposomal lipids and release of theencapsulated agents. In the present context, the vaccine conjugate inthe composition according to the invention can be localized on thesurface of the liposome, to facilitate antigen presentation withoutdisruption of the liposome or endocytosis. Irrespective of the mechanismor delivery, however, the result is the intracellular disposition of theassociated vaccine conjugate.

Liposomal vectors may be anionic or cationic. Anionic liposomal vectorsinclude pH sensitive liposomes which disrupt or fuse with the endosomalmembrane following endocytosis and endosome acidification.

Other suitable liposomes that are used in the compositions and methodsof the invention include multilamellar vesicles (MLV), oligolamellarvesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles(SUV), medium-sized unilamellar vesicles (MIN), large unilamellarvesicles (LUV), giant unilamellar vesicles (GUV), multivesicularvesicles (MVV), single or oligolamellar vesicles made by reverse-phaseevaporation method (REV), multilamellar vesicles made by thereverse-phase evaporation method (MLV-REV), stable plurilamellarvesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared byextrusion methods (VET), vesicles prepared by French press (FPV),vesicles prepared by fusion (FUV), dehydration-rehydration vesicles(DRV), and bubblesomes (BSV). Techniques for preparing these liposomesare well known in the art.

Other forms of delivery particle, for example, microspheres and thelike, also are contemplated for delivery of the vaccine conjugate.

In one embodiment, the vaccine composition may be included in an animalfeed (i.e., a foodstuff suitable for consumption by an animal)comprising a composition and/or a vaccine conjugate according to theinvention. This may, in non-limiting examples, be in the form ofpellets, crumbs or a mash which may further comprise, again for exampleonly, grain, grass and/or protein components. The composition may alsobe included in drinking liquids and/or administered via a spray into theatmosphere surrounding the animal which is, consequently, inhaled by theanimal.

The method and diagnostic conjugate according to the aspects of theinvention described above enable such useful vaccine conjugates andcompositions to be prepared for use to vaccinate animals againstinfection by Brucella, based on synthetic, exclusively -(1-2)- linked,4,6-dideoxy-4-acylamido-α-pyranose oligosaccharides. This is because themethod according to the invention provides a DIVA test to distinguishanimals vaccinated with such a vaccine from animals infected fromBrucella.

Therefore, the vaccine conjugate and/or vaccine composition according tothe invention may be for use in a method of vaccinating an animalagainst infection by Brucella, the method comprising administering thevaccine conjugate and/or vaccine composition to the animal. An eighthaspect of the invention, therefore, provides a method of vaccinating ananimal against infection by Brucella, the method comprisingadministering the vaccine conjugate and/or vaccine composition to theanimal. The method may further comprise subsequently confirming thepresence, in a biological sample obtained from the animal, of anti-Aand/or anti-C/Y antibodies, protective against Brucella infection, bymeans of a method according to the first aspect of the invention. Forexample, this may be by contacting the sample with a diagnosticconjugate which is a universal antigen as defined herein.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to” and donot exclude other moieties, additives, components, integers or steps.Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Other features of the present invention will become apparent from thefollowing examples. Generally speaking, the invention extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including the accompanying claims and drawings). Thus,features, integers, characteristics, compounds or chemical moietiesdescribed in conjunction with a particular aspect, embodiment or exampleof the invention are to be understood to be applicable to any otheraspect, embodiment or example described herein, unless incompatibletherewith.

Moreover, unless stated otherwise, any feature disclosed herein may bereplaced by an alternative feature serving the same or a similarpurpose.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention will now be described, by way of exampleonly, with reference to FIGS. 1-27 in which:

FIG. 1 shows target pentasaccharide (1) which exhibits preferred bindingto M-specific antibodies and nonasaccharide (2) designed to bind both A-and M-specific antibodies;

FIG. 2 shows results (expressed as a percentage of a common positivecontrol) from the B. melitensis 16M OPS iELISA (x-axis) and Y.enterocolitica O:9 OPS iELISA (y-axis); the solid diamonds represent theresults for sera from cattle confirmed as infected with B. abortusbiovar 1 (n=45) and the crosses represent the results for sera fromnon-Brucella infected cattle that are positive in one or moreconventional serodiagnostic assays (n=68);

FIG. 3A shows results (expressed as a percentage of a common positivecontrol) for B. melitensis 16M OPS iELISA (x-axis) and Y. enterocoliticaO:9 OPS iELISA (y-axis) ELISAs; the solid diamonds represent the resultsfor sera from swine confirmed as infected with B. suis biovar 1 (n=41)and the crosses represent the results for sera from non-Brucellainfected swine from herds with one or more animals that are positive inone or more conventional serodiagnostic assays (n=52);

FIG. 3B shows results (expressed as a percentage of a common positivecontrol) for B. abortus S99 OPS iELISA (x-axis) and Y. enterocoliticaO:9 OPS iELISA (y-axis) ELISAs; the solid diamonds represent the resultsfor sera from swine confirmed as infected with B. suis biovar 1 (n=41)and the crosses represent the results for sera from non-Brucellainfected swine from herds with one or more animals that are positive inone or more conventional serodiagnostic assays (n=52);

FIGS. 4A-4D show selected ion count for the tetrasaccharide (m/z=711.3)(A) from B. melitensis 16M core OPS (three significant peaks visible at8:05, 8.50 & 11:10 mins:secs), (B) from B. abortus S99 core OPS (fivesignificant peaks visible at 8:05, 8:50, 9:20, 10:05 and 10:40mins:secs), (C) from Y. enterocolitica O:9 core OPS (six significantpeaks visible at 8:05, 9:20, 9:55, 10:10, 10:40 and 11:30 mins:secs);(D) from B. abortus S99 core OPS eluted from an affinity chromatographycolumn conjugated with anti-Brucella mAb BM40 (three significant peaksvisible at 8:50, 10:30 and 11:15 mins:secs); all core OPS hydrolysed andpurified (by size exclusion chromatography) and analysed on agraphitised carbon HPLC column online with ESI-triple quadrupole massspectrometer;

FIG. 5 shows inhibition of BM40 mAb binding to solid phase B. melitensis16M OPS antigen (y-axis) by varied concentrations of competing liquidphase antigen (x-axis), with three types of competing OPS antigens (asshown in legend) evaluated in addition to the TSM antigen according tothe invention;

FIGS. 6A-6B show inhibition of rabbit anti-‘M’ monospecific antiserabinding to solid phase B. melitensis 16M core-OPS antigen (y-axis) (FIG.6A) or inhibition of rabbit anti-′A′ monospecific antisera binding tosolid phase B. abortus S99 core-OPS antigen (y-axis) (FIG. 6B) by variedconcentrations of competing liquid phase antigen (x-axis), with threetypes of competing OPS antigens evaluated (as shown in legend) inaddition to the TSM (tetrasaccharide) antigen according to theinvention;

FIG. 7 shows the results (expressed as a percentage of a common positivecontrol) from the B. melitensis 16M OPS iELISA (x-axis) and TSM antigeniELISA (y-axis); the solid diamonds represent the results for sera fromcattle confirmed as infected with B. abortus biovar 1 (n=45) and thecrosses represent the results for sera from non-Brucella infected cattlethat are positive in one or more conventional serodiagnostic assays(n=68);

FIG. 8 shows ELISA titration curves for Brucella A- and M-specific mAbs,YsT9-1 and Bm10; pentasaccharide conjugate 42 (right panel) andnona-saccharide conjugate 43 (left panel);

FIG. 9 shows results (expressed as a percentage of a common positivecontrol) from the BSA-nonasaccharide conjugate iELISA (x-axis) andBSA-pentasaccharide conjugate iELISA (y-axis); the solid diamondsrepresent the results for sera from cattle confirmed as infected with B.abortus biovar 1 (n=45) and the crosses represent the results for serafrom non-Brucella infected cattle that are positive in one or moreconventional serodiagnostic assays (n=68);

FIG. 10 shows results (expressed as a percentage of a common positivecontrol) from the BSA-tetrasaccharide conjugate iELISA (x-axis) andBSA-disaccharide conjugate iELISA (y-axis); the solid diamonds representthe results for sera from cattle confirmed as infected with B. abortusbiovar 1 (n=45) and the crosses represent the results for sera fromnon-Brucella infected cattle that are positive in one or moreconventional serodiagnostic assays (n=68);

FIG. 11 shows results (expressed as a percentage of a common positivecontrol) from the BSA-trisaccharide conjugate (with terminal α-1,3 link)iELISA (x-axis) and BSA-trisaccharide conjugate (with terminal α-1,2link) iELISA (y-axis); the solid diamonds represent the results for serafrom cattle confirmed as infected with B. abortus biovar 1 (n=45) andthe crosses represent the results for sera from non-Brucella infectedcattle that are positive in one or more conventional serodiagnosticassays (n=68);

FIG. 12 shows ROC Curves generated from the results of the Y.enterocolitica O:9 OPS, B. melitensis 16M OPS, BSA-pentasaccharide,BSA-tetrasaccharide and BSA-disaccharide conjugate iELISAs as applied tosera from sera from cattle confirmed as infected with B. abortus biovar1 (n=45) and sera from non-Brucella infected cattle that are positive inone or more conventional serodiagnostic assays (n=68);

FIG. 13 shows the results from the B. melitensis 16M OPS iELISA asapplied to sera from four cattle experimentally infected with Brucellaabortus strain 544 (Brucella #1 to #4, solid lines) and four cattleexperimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4,dashed lines); samples were taken on weeks 3, 7, 16 and 24 postinfection;

FIG. 14 shows the results from the BSA-nonasaccharide conjugate iELISAas applied to sera from four cattle experimentally infected withBrucella abortus strain 544 (Brucella #1 to #4, solid lines) and fourcattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to#4, dashed lines); samples were collected, on weeks 3, 7, 16 and 24 postinfection;

FIG. 15 shows the results from the BSA-pentasaccharide conjugate iELISAas applied to sera from four cattle experimentally infected withBrucella abortus strain 544 (Brucella #1 to #4, solid lines) and fourcattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to#4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 postinfection;

FIG. 16 shows the results from the B. melitensis 16M OPS iELISA (x-axis)and the BSA-pentasaccharide conjugate iELISA (y-axis) as applied to sera(n=16) from four cattle experimentally infected with Brucella abortusstrain 544 (solid diamonds), and sera (n=16) from four cattleexperimentally infected with Y. enterocolitica O:9 (crosses). Sampleswere taken on weeks 3, 7, 16 and 24 post infection; the scatter plotonly distinguishes between samples from different infection types, notby animal and time post infection;

FIG. 17 shows the results from the BSA-tetrasaccharide conjugate iELISAas applied to sera from four cattle experimentally infected withBrucella abortus strain 544 (Brucella #1 to #4, solid lines) and fourcattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to#4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 postinfection;

FIG. 18 shows the results from the BSA-disaccharide conjugate iELISA asapplied to sera from four cattle experimentally infected with Brucellaabortus strain 544 (Brucella #1 to #4, solid lines) and four cattleexperimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4,dashed lines); samples were taken on weeks 3, 7, 16 and 24 postinfection;

FIG. 19 shows the results from the BSA-tetrasaccharide conjugate iELISA(x-axis) and the BSA-disaccharide conjugate iELISA (y-axis) as appliedto sera (n=16) from four cattle experimentally infected with Brucellaabortus strain 544 (solid diamonds), and sera (n=16) from four cattleexperimentally infected with Y. enterocolitica O:9 (crosses). Sampleswere taken on weeks 3, 7, 16 and 24 post infection; the scatter plotonly distinguishes between samples from different infection types, notby animal and time post infection;

FIG. 20 shows results (expressed as a percentage of a common positivecontrol) from the BSA-nonasaccharide conjugate iELISA (x-axis) andBSA-pentasaccharide conjugate iELISA (y-axis); the solid diamondsrepresent the results for sera from cattle confirmed as infected with B.abortus biovar 1 (n=45) and the crosses represent the results for serafrom randomly sampled brucellosis free cattle (n=125);

FIG. 21 shows results (expressed as a percentage of a common positivecontrol) from the BSA-tetrasaccharide conjugate iELISA (x-axis) andBSA-disaccharide conjugate iELISA (y-axis); the solid diamonds representthe results for sera from cattle confirmed as infected with B. abortusbiovar 1 (n=45) and the crosses represent the results for sera fromrandomly sampled brucellosis free cattle (n=125);

FIG. 22 shows the results (expressed as a percentage of a commonpositive control) for B. melitensis 16M OPS iELISA (x-axis) and TSMantigen (y-axis) ELISAs; the solid diamonds represent the results forsera from sheep and goats infected with B. melitensis biovar 3 (n=61)and the open triangles represent the results for sera from non-Brucellainfected sheep and goats that have been randomly sampled within GreatBritain (n=94);

FIG. 23 shows the results (expressed as a percentage of a commonpositive control) for the BSA-pentasaccharide conjugate iELISA (x-axis)and BSA-nonasaccharide conjugate (y-axis) ELISAs; the solid diamondsrepresent the results for sera from sheep and goats infected with B.melitensis biovar 3 (n=61) and the open triangles represent the resultsfor sera from non-Brucella infected sheep and goats that have beenrandomly sampled within Great Britain (n=94);

FIG. 24 shows titration of human serum (#2) from a human patientdiagnosed Brucella suis positive by bacterial culture, againstcopovidone-conjugated disaccharide conjugates 99a and 99b;

FIG. 25 shows titration of monoclonal antibodies and human serum #2against ELISA plates coated with low loading, “Brucella A type”hexasaccharide conjugate 100b;

FIG. 26 shows titration of monoclonal antibodies and human serum #2against ELISA plates coated with universal antigen “Brucella A and Mtype” hexasaccharide conjugate 98; and

FIG. 27 shows the binding of HRP conjugated BM40 anti-M mAb, expressedas iELISA optical density (OD) on the y-axis, against threeBSA-oligosaccharide conjugates shown as three separate lines: thedisaccharide, a trisaccharide and the tetrasaccharide

EXAMPLES Oligosaccharide and Conjugate Synthesis

General methods are provided in the section below headed “GeneralSynthesis Methods”.

Pentasaccharide 1 (FIG. 1; e.g. Formula XI) was selected as the largestsized antigen that might selectively exhibit M-type characteristics withlimited cross reaction with A-specific antibodies. Nonasaccharide 2(Formula XV above) was considered to be an antigen that would containtwo A- and one M-type epitopes, which would serve as a universal antigento detect antibodies in animals or humans infected by B. abortus, B.melitensis and B. suis.

The large size of the target oligosaccharides 1 and 2, the incorporationof an internal 1,3 linkage and a tether for antigen synthesis was notpreviously attempted (Peters & Bundle (1989) Can. J. Chem. 67, 497-502)and necessitated the development of an improved synthetic strategy. Thelinker 5-methoxycarbonylpentanol (Lemieux et al. (1975) J. Am. Chem.Soc. 97, 4076-4083; Ogawa et al. (1996) Carbohydr. Res. 293, 173-94) waschosen for its compatibility with the strategy employed to deprotect theassembled oligosaccharides and for its flexibility in offering severalroutes for subsequent conjugation to protein to create glycoconjugateantigens (Kamath et al. (1996) Glycoconjugate 13, 315-319). Two distinctstrategies were employed to synthesis first the penta- andnonasaccharides, with a second revised strategy to synthesise di-, tri-,tetra- and hexasaccharides. The second strategy employed related, butslightly modified, protection schemes for the construction of themonosaccharide and disaccharide synthons.

Synthetic Strategy 1

Well-established methods were utilised to synthesize compounds 3-6, asoutlined below (Bundle et al. (1998) Carbohydr. Res. 174, 239-251).Lewis acid catalyzed glycosidation of 6 afforded the allyl glycoside 7,which was transesterified to 8. Formation of a 2,3-orthoester derivativewhich undergoes regioselective opening afforded the selectivelyprotected building block 9 (Scheme 1).

We used N-phenyl trifluoroacetimidates for glycosylation reactions sincethis donor has been shown to be more efficient than the correspondingtrichloroacetimidate derivative for glycosylations involving 6-deoxysugar donors (Hanashima et al. (2007) Org. Lett. 9, 1777-1779).Selective deacetylation of 5 gave 10, and then reaction with theN-phenyl trifluoroacetimidoyl chloride in presence of cesium carbonateas base gave the glycosyl N-phenyl trifluoroacetimidate donor 11(Hanashima et al. (2007) Org. Lett. 9, 1777-1779). Monosaccharide 5 wasalso converted to thioglycoside donor 12 and transesterificationafforded the acceptor 13 (Peters & Bundle (1989) Can. J. Chem. 67,491-496). Glycosylation of 13 by 11 was performed in the presence oftrimethylsilyl trifluoromethanesulfonate to give disaccharide 14 in 94%yield with complete stereocontrol and without detectable amounts ofβ-anomer (Scheme 2).

The 1,3-linked trisaccharide building block 15 was created as its allylglycoside since the selective removal of this anomeric protecting groupallows facile access to a hemiacetal and subsequently an imidate leavinggroup (Du et al. (2001) Tetrahedron 57, 1757-1763). Glycosylationreactions were tried with allyl as a leaving group but all attempts todo so failed (Wang et al. (2007) J. Org. Chem. 72, 5870-2873).Consequently, 15 was selectively deprotected with palladium chloride inacetic acid (Du et al. (2001) Tetrahedron 57, 1757-1763) to givehemiacetal 16 which was in turn converted to the N-phenyltrifluoroacetimidate donor 17 (Scheme 3).

Glycosylation of 5-methoxycarbonylpentanol by thioglycoside 14 gavemoderate to poor yields due to the low reactivity of acceptors of thistype (Lemieux et al. (1975) J. Am. Chem. Soc. 97, 4076-4083). Hydrolysisof the thioethyl glycoside 14 gave hemiacetal 18 which was converted itto imidate 19. The six carbon linker 20 (El Fangour et al. (2004) J.Org. Chem. 69, 2498-1503) which was glycosylated by 19 to give theprotected disaccharide glycoside 21 (Hou & Ková{hacek over (c)} (2010)Carbohydr. Res. 345, 999-1007). Transesterification of 21 gave thetether glycoside acceptor 22 (Scheme 4).

Pentasaccharide 23 was obtained from building blocks trisaccharide 17and disaccharide glycoside 22 in 68% yield using TMSOTf as the activator(Scheme 5). Stepwise deprotection followed the sequence: deacetylationto give 24 in quantitative yield, azido group reduction with hydrogensulfide to give 25. Compound 25 was directly formylated by a mixedanhydride (acetic anhydride/formic acid 2:1) to give 26 (Bundle et al.(1988) Carbohydr. Res. 174, 239-251). Following introduction of theN-formamido groups, NMR analyses of all subsequent compounds becamedifficult due to the presence of E/Z rotamers for each formyl group,leading to a potential mixture of 32 isomers. Their identity wasconfirmed by a limited set of characteristic NMR resonances and highresolution mass measurements. Pentasaccharide 1 was obtained byhydrogenolysis of benzyl ethers.

The synthesis of nonasaccharide 2 was envisaged as the creation of apentasaccharide donor terminated by a 1,3 linkage which would then allowfor a pentasaccharide donor with a participating group at C-2 to guidethe stereoselective α-glycosylation of an exclusively 1,2-linkedtetrasaccharide. To achieve the synthesis of the pentasaccharide donor,compound 14 was deprotected to give the corresponding acceptor 27 whichwas glycosylated by imidate donor 19. Tetrasaccharide 28 was formed intoluene at 100° C. as described for 21 (Hou & Ková{hacek over (c)}(2010) Carbohydr. Res. 345, 999-1007). Tetrasaccharide thioglycoside 28was used directly as the donor for glycosylation of the monosaccharideglycoside 9 to give the α1,3-linkage. The allyl group of pentasaccharide29 was then removed (Du et al. (2001) Tetrahedron 57, 1757-1763) to give30 and the imidate donor 31 was obtained following reaction withN-phenyl trifluoroacetimidoyl chloride (Scheme 6).

Tetrasaccharide tether glycoside 32 was obtained by a 2+2 glycosylationof disaccharide acceptor 22 by the disaccharide donor 19 employing thesame condition used to prepare 28. Transesterification of 32 gave thetetrasaccharide acceptor 33 which was glycosylated by pentasaccharidedonor 31 to give nonasaccharide 34 in 30% yield. The sequence ofdeprotection steps (deacetylation to 35, reduction of azide to 36,N-formylation to 37 and hydrogenation) to give 2 followed the order usedto obtain pentasaccharide 1 (Scheme 7).

The final compounds 1 and 2 were purified by reverse phase HPLC. FullNMR assignments were performed on the azido penta and nonasaccharidederivatives 24 and 35. Selected characteristic NMR resonances and highresolution mass confirmed the identity of derivatives 26 and 37 and thetarget oligosaccharides 1 and 2.

To enable conjugation to protein, pentasaccharide and nonasaccharideglycosides 1 and 2 were first converted to the respective amides 38 and39 by reaction with ethylenediamine (Scheme 8). Reaction of 38 and 39with dibutyl squarate gave the squarate half esters 40 and 41 which wereisolated by reverse phase HPLC. The corresponding pentasaccharide andnonasaccharide bovine serum albumin (BSA) glycoconjugates 42 and 43 wereprepared by reaction of a twenty to one molar ratio of 40 and 41 withBSA in borate buffer for 3 days. MALDI-TOF mass spectrometry indicatedthat each conjugate contained approximately 16 copies of theoligosaccharides per molecule of BSA.

Synthetic Strategy 2

This strategy set out to arrive at shorter oligosaccharides 44-47 thatwould provide M-specific antigens and achieve the level ofdiscrimination described above.

Two additional compounds were synthesized; hexasaccharide 48 to providea pure A epitope and compound 49 which provides an oligosaccharide ofminimal size that encompasses both A and M epitopes. Oligosaccharide 49,along with the nonasaccharide 2, is a universal Brucella antigen. Itcorresponds to the terminal hexasaccharide of the Brucella O-antigendisclosed by Kubler-Kielb and Vinogradov (Carbohydr. Res. (2013) 378,144-147).

In contrast to the synthesis of 1 and 2 where acetate esters and benzylethers were used, the synthesis of oligosaccharides 44-49 made use ofbenzoate esters and benzyl ether protecting groups to allow for theconstruction of 1,2 and 1,3 linkages. This distinct difference inprotecting group strategy from the earlier synthesis of 1 and 2 allowedthe use of trichloroacetimidate donors rather than the more difficult toprepare N-phenyl trifluoroacetimidates 11, 19 and 31. The synthons usedto make oligosaccharides 44-49 were compounds 50-66. The monosaccharideimidates 53, 58 and 62 were synthesized as shown (Schemes 9 and 10). Twodisaccharide thioglycosides 63 and 64 were prepared by glycosylation of13 by imidates 53 and 62 (Scheme 10). The two 5-methoxycarbonylpentanolglycosides 65 and 66 were prepared by literature methods (Saksena et al.(2008) Carbohydr. Res. 343, 1693-1706; Saksena et al. (2005)Tetrahedron: Asymmetry 16, 187-197). Imidate 62 was used in thesynthesis of 48 and 49.

The 1,3 linked disaccharide 67 was obtained by reaction of the imidate53 with the acceptor 65. Trisaccharide 68 was obtained by reaction ofthe disaccharide thioglycoside 63 with the monosaccharide glycoside 65.Reaction of the monosaccharide imidate 58 with 66 gave the 1,2 linkeddisaccharide 69. When 69 was de-O-benzoylated the resulting disaccharide70 could be glycosylated by the monosaccharide imidate 53 to give thetrisaccharide 71 with a terminal 1,3 linkage. Disaccharide 70 alsoprovides access to tetrasaccharide 72 when it was reacted withdisaccharide thioglycoside 63 (Scheme 11).

Hexasaccharides 48 and 49 were elaborated on the monosaccharideglycoside 66 by glycosylation with the disaccharide thioglycoside 64 togive the trisaccharide 73 which after transesterification provides thealcohol acceptor 74, which serves as the common trisaccharideintermediate leading to both hexsaccharides (Scheme 12). The exclusively1,2-linked hexasaccharide 48 was prepared by glycosylation of 74 by thedisaccharide thioglycoside 64 to give pentasaccharide 75.Transesterification of the terminal benzoate group gave thepentasaccharide alcohol 76, which afforded hexasaccharide 77 afterglycosylation by the imidate 53. Glycosylation of trisaccharide alcohol74 by imidate 58 afforded tetrasaccharide 78. After transesterication of78 the tetrasaccharide alcohol 79 is set up for introduction of a1,3-linkage. Reaction with the disaccharide thioglycoside 63 gives theprotected hexasaccharide 80.

Deprotection of the six oligosaccharides 67, 68, 71, 72, 77 and 80employed identical reaction conditions (Scheme 13). This involvedtransesterification to remove benzoate esters, reduction of the azidegroups to amines, acylation of amines by formic anhydride to affordformamides and lastly catalytic hydrogenation to afford the targetoligosaccharides as 5 methoxycarbonylpentanol glycosides 44-49.

Conversion of the 5 methoxycarbonylpentanol glycosides 44-49 to antigensfor diagnostic or vaccine applications followed a similar protocol tothat described for the penta and nonasaccharides (Scheme 14).

A non-protein polymer aminated co-povidone was also used as analternative and potential superior antigen for immunoassays.Representative conjugations are described for the disaccharide 44 andthe hexasaccharide 48 (Scheme 15).

Potential vaccine candidates were synthesized by conjugating the twohexasaccharides 48 and 49 to monomeric tetanus toxoid. The squarate halfesters 91 and 92 were each conjugated to tetanus toxoid to provideantigens 101 and 102 (Scheme 16).

General Synthesis Methods

Analytical TLC was performed on Silica Gel 60-F₂₅₄ (Merck, Darmstadt)with detection by quenching of fluorescence and/or by charring with 5%sulfuric acid in ethanol. All commercial reagents were used as supplied.Column chromatography was performed on Silica Gel 230-400 mesh, 60 Å(Silicycle, Ontario) with HPLC quality solvents. Molecular sieves werecrushed and stored in an oven at 150° C. and flamed dried under vacuumbefore use. Organic solutions were dried with anhydrous MgSO₄ prior toconcentration under vacuum at <40° C. (bath). All final compounds werepurified by reverse phase chromatography performed on a Waters 600 HPLCsystem, using a Beckmann semi-preparative C-18 column (10×250 mm, 5μ)with a combination of acetonitrile and water as eluents. Products weredetected with a Waters 2487 UV detector.

Optical rotations were measured with a Perkin-Elmer 241 polarimeter forsamples in a 10 cm cell at 21±2° C. [α]_(D) values are given in units of10⁻¹ deg cm² g⁻¹, with [α]_(D) ²⁰ indicating that the temperature was20° C. and [α]_(D) ²¹ indicating that the temperature was 21° C.

¹H NMR spectra were recorded on 500, 600 or 700 MHz spectrometers. Firstorder proton chemical shifts δ_(H) are referenced to either residualCHCl₃ (δ_(H) 7.27, CDCl₃) or CD₂HOD (δ_(H) 3.30, CD₃OD), or internalacetone (δ_(H) 2.225, D₂O). ¹³C NMR spectra were recorded at 125 MHz,and chemical shifts are referenced to internal CDCl3 (δ 77.23) orexternal acetone (δ 31.07). The assignment of resonances for allcompounds was made by two-dimensional homonuclear and heteronuclearchemical shift correlation experiments. Mass analysis was performed bypositive-mode electrospray ionization on a hybrid sector-TOF massspectrometer and for protein glycoconjugates by MALDI mass analysis,employing sinapinic acid as matrix.

The numbering used for compounds 4-41 is as follows:

and for compounds 42-92:

Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (4)

A solution of 3 (Bundle et al. (1988) Carbohydr. Res. 174, 239-251)(1.09 g, 5.36 mmol) and Bu₂SnO (1.5 g, 6 mmol) in toluene (50 mL) wasstirred at 140° C. for 2 h. Then, after cooling down, benzyl bromide(0.7 mL, 5.9 mmol) and Bu₄NBr (1.9 g, 5.9 mmol) were added and themixture was stirred overnight at 65° C. After evaporation of thesolvent, a purification on a silica gel column (hexane/ethyl acetate8:1) gave pure 4 (1.38 g, 87%). The NMR parameters are in agreement withthe literature (Boschiroli et al. (2001) Curr. Op. Microbiol. 4, 58-64):¹H NMR (500 MHz, CDCl₃): δ 7.5-7.3 (m, 5H; H—Ar), 4.72 (d, ³J_(1,2)=1.7Hz, 1H; H-1), 3.98 (dt, ³J_(2,3)=3.3 Hz, ³J_(2,OH)=1.6 Hz, 1H; H-2),3.72 (dd, ³J_(3,4)=9.6 Hz, 1H; H-3), 3.36 (s, 3H; OCH₃), 2.39 (d, 1H;OH), 1.44 ppm (d, 3H; H-6); ¹³C NMR (126 MHz, CDCl₃): δ 137.2, 128-129,100.0, 78.3, 72, 67.2, 66.4, 63.9, 55.0, 18.4 ppm; HRMS (ESI): m/z calcdfor C₁₄H₁₉N₃NaO₄ [M+Na]⁺: 316.12678. found: 316.12705.

1,2-Di-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (5)

A solution of 4 (1.03 g, 5.07 mmol) in acetic anhydride/aceticacid/sulfuric acid (50:20:0.5, 35 mL) was stirred for 1.5 h at roomtemperature, and then poured into ice-cold 1 M K₂CO₃ solution. Theproduct was extracted with dichloromethane and the extract was driedover MgSO₄. The solvent was evaporated and co-evaporated with toluene. Achromatography column on silica (hexane/ethyl acetate 10:1) gave 5 as ananomeric mixture (1.4 g, 82%, α/β 93:7). The NMR parameters are inagreement with the literature (Boschiroli et al. (2001) Curr. Op.Microbiol. 4, 58-64): ¹H NMR (500 MHz, CDCl₃): δ(α) 7.4-7.3 (m, 5H;H—Ar), 6.02 (d, ³J_(1,2)=2 Hz, 1H; H-1), 5.34 (dd, ³J_(2,3)=3.3 Hz, 1H;H-2), 2.15, 2.11 (2s, 6H; OAc), 1.34 ppm (d, 3H; H-6); ¹³C NMR (126 MHz,CDCl₃): δ 169.8, 168.3, 137.0, 128-129, 91.0, 75.8, 71.8, 69.3, 66.3,63.6, 20.9, 20.8, 18.5 ppm; elemental analysis calcd (%) for C₁₇H₂₁N₃O₆:C, 56.2, H, 5.8, N, 11.7. found: C, 56.2, H, 5.7, N, 11.3.

1,2,3-Tri-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (6)

Compound 3 (1.03 g, 5.07 mmol) was acetylated with the same protocolused to obtain compound 5. Compound 6 (1.31 g, 82%) was obtained in aα/β 95:5 mixture. The separation was done only for analysis: The NMRparameters and physical constants are in agreement with the literature(Bundle et al. (1988) Carbohydr. Res. 174, 239-251): [α]_(D) ²⁰(α)=+122(c=1.2 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ(α) 5.98 (d, ³J_(1,2)=1.6Hz, 1H; H-1), 2.15, 2.13, 2.07 (3s, 9H; OAc), 1.36 ppm (d, 3H; H-6); ¹³CNMR (126 MHz, CDCl₃): δ 169.7, 169.6, 168.3, 90.6, 70.1, 69.3, 67.8,62.1, 20.9, 20.7, 20.7, 18.4 ppm; HRMS (ESI): m/z calcd for C₁₂H₁₇N₃NaO₇[M+Na]⁺: 338.09587. found: 338.09559; elemental analysis calcd (%) forC₁₂H₁₇N₃O₇: C, 45.7, H, 5.4, N, 13.3. found: C, 45.35, H, 5.45, N, 13.2.

Allyl 2, 3-di-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (7)

To a solution of 6 (900 mg, 2.85 mmol) in dichloromethane (4 mL), wasadded BF₃.Et₂O (0.4 mL, 3.24 mmol). The mixture was stirred for 1 h atroom temperature before adding allylic alcohol (0.3 mL, 4.41 mmol) andthen stirred again for 2 days. Once the reaction was done, the mixturewas cooled to 0° C., a satd. NaHCO₃ solution was added and the mixturewas stirred for 30 min. The product was extracted with ethyl acetate,the extract was dried (MgSO₄), filtrated and concentrated. Purificationon column chromatography (hexane/ethyl acetate 10:1) gave the allylglycoside 7 (625 mg, 70%): [α]_(D) ²⁰=+103 (c=1.5 in CHCl₃); ¹H NMR (500MHz, CDCl₃): δ 5.25 (dd, ³J_(2,3)=3.6 Hz, ³J_(3,4)=10 Hz, 1H; H-3), 5.23(dd, ³J_(1,2)=1.7 Hz, 1H; H-2), 4.76 (d, ³J_(1,2)=1.7 Hz, 1H; H-1),2.15, 2.08 (2s, 6H; OAc), 1.37 ppm (d, 3H; H-6); ¹³C NMR (126 MHz,CDCl₃): δ 169.9, 169.6, 133.1, 118.1, 96.5, 70.4, 69.2, 68.4, 67.0,62.7, 20.9, 20.8, 18.3 ppm; elemental analysis calcd (%) for C₁₃H₁₉N₃O₆:C, 49.8, H, 6.1, N, 13.4. found: C, 49.9, H, 6.3, N, 13.5.

Allyl 4-azido-4,6-dideoxy-α-D-mannopyranoside (8)

Allyl glycoside 7 (625 mg, 2 mmol) in methanol (20 mL) was treated witha 0.1 M solution of sodium methoxide (0.3 mL). After 1 h, the reactionwas complete and neutralized with ion exchange resin H⁺. Filtration andremoval of the solvent under vacuum gave pure diol 8 (447 mg, 98%):[α]_(D) ²⁰=+117 (c=1.1 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 4.84 (d,³J_(1,2)=1.7 Hz, 1H; H-1), 3.93 (dd, ³J_(2,3)=3.4 Hz, 1H; H-2), 1.34 ppm(d, 3H; H-6); ¹³C NMR (126 MHz, CDCl₃): δ 133.4, 117.7, 98.5, 70.5,70.2, 68.2, 66.8, 66.0, 18.3 ppm (C-6); HRMS (ESI): m/z calcd forC₉H₁₅N₃NaO₄ [M+Na]⁺: 252.09548. found: 252.09579.

Allyl 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (9)

To a solution of diol 8 (77 mg, 0.34 mmol) in dichloromethane (3.5 mL)were added triethyl orthoacetate (0.6 mL, 3.3 mmol) andp-toluenesulfonic acid (5 mg, 0.03 mmol). The mixture was stirred for 3hrs at 50° C. When complete, the reaction was neutralized bytriethylamine and concentrated. Acetic acid (80%, 8 mL) was added andthe mixture was stirred for 30 min at room temperature, thenconcentrated. Compound 9 (91 mg, 98%) was purified by chromatography onsilica (ethyl acetate/hexanes 1:10): [α]_(D) ²⁰=+78 (c=1.0 in CHCl₃); ¹HNMR (500 MHz, CDCl₃): δ 5.08 (dd, ³J_(1,2)=1.7 Hz, ³J_(2,3)=3.6 Hz, 1H;H-2), 4.80 (d, 1H; H-1), 2.15 (s, 3H; OAc), 1.35 ppm (d, 3H; H-6); ¹³CNMR (126 MHz, CDCl₃): δ 170.8, 133.2, 117.9, 96.5, 71.7, 69.2, 68.3,66.9, 66.0, 21.0, 18.3 ppm; elemental analysis calcd (%) for C₁₁H₁₇N₃O₅:C, 48.7, H, 6.3, N, 15.5. found: C, 48.8, H, 6.2, N, 15.3.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranose (10)

To a solution of 5 (484 mg, 1.33 mmol) in dichloromethane (3 mL),dimethylamine (2 M in THF, 1.8 mL, 3.6 mmol) was added dropwise. Thesolution was then stirred for 2 days at room temperature. Afterevaporation of the solvent, pure compound 10 (432 mg) was obtainedquantitatively: ¹H NMR (500 MHz, CDCl₃): δ 7.2-7.4 (m, 5H; H—Ar), 5.30(s, 1H; H-1 β), 5.17 (d, 1H; H-1 α), 2.19 (s, 3H; OAc β), 2.13 (s, 3H;OAc α), 1.38 (d, 3H; H-6 β), 1.33 ppm (d, 3H; H-6 α); ¹³C NMR (126 MHz,CDCl₃): δ 170.8, 170.3, 137.2, 136.8, 128-129, 92.9, 92.5, 78.5, 75.6,71.7, 71.6, 71.2 (C-5 β), 68.5, 67.7, 67.0, 64.0, 63.4, 21.0, 20.9,18.65, 18.5 ppm (C-6 β); elemental analysis calcd (%) for C₁₅H₁₉N₃O₅: C,56.1, H, 6.0, N, 13.1. found: C, 56.0, H, 6.0, N, 13.0.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosylN-phenyltrifluoroacetimidate (11)

Compound 10 (1.04 g, 3.2 mmol) was dissolved in dry dichloromethane (30mL). N-phenyl trifluoroacetimidoyl chloride (1.2 mL, 9.6 mmol) andCs₂CO₃ (3.2 g, 9.6 mmol) were added and the mixture was stirredovernight at room temperature. After filtration through celite, compound11 (1.4 g, 87%) was purify on silica gel column (hexane/ethyl acetate10:1): ¹H NMR (500 MHz, CDCl₃): δ 7.4-6.8 (m, 10H; H—Ar), 6.2 (br s, 1H;H-1 α), 5.85 (br s, 1H; H-2 α), 5.8 (br s, 1H; H-1 β), 5.46 (br s, 1H;H-2 β), 2.2 (s, 3H; OAc β), 2.15 (s, 3H; OAc α), 1.44 (d, ³J_(5,6)=6.0Hz, 3H; H-6 α), 1.33 ppm (d, ³J_(5,6)=6.1 Hz, 3H; H-6 β); HRMS (ESI):m/z calcd for C₂₃H₂₃F₃N₄NaO₅ [M+Na]⁺: 515.15128. found: 515.15147.

Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (14)

Donor 11 (3.44 g, 7 mmol) and acceptor 13 (obtained from 5 via 12)(Peters & Bundle (1989) Can. J. Chem. 67, 491-496) (1.75 g, 5.4 mmol)were dissolved in dry dichloromethane (50 mL) and TMSOTf (0.1 mL, 0.55mol) was added at 0° C. The reaction was complete after 30 min. ofstirring at 0° C., then 30 min. at room temperature and was quenchedwith few drops of NEt₃. Disaccharide 14 (3.19 g, 94%) was obtained pureafter flash chromatography column (toluene/ethyl acetate 1:0, then 9:1).The NMR parameters and physical constants are in agreement with theliterature (Crump et al. (2003) Emerg. Infect. Dis. 9, 539-544): [α]_(D)²⁰=+129 (c=1.2 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.4-7.2 (m, 10H;H—Ar), 5.39 (dd, ³J_(1,2)=1.6 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(II)), 5.16(d, ³J_(1,2)=1.1 Hz, 1H; H-1^(I)), 4.81 (d, 1H; H-1^(II)), 3.89 (dd,³J_(2,3)=2.9 Hz, 1H; H-2^(I)), 2.53 (2qd, ²J=13 Hz, ³J=7.4 Hz, 2H;S—CH₂—CH₃), 2.10 (s, 3H; Ac), 1.30 (d, 3H; H-6^(II)), 1.29 (d, 3H;H-6^(I)), 1.25 ppm (t, 3H; S—CH₂—CH₃); ¹³C NMR (125 MHz, CDCl₃): δ170.0, 137.5, 137.2, 129-128, 99.7, 83.4, 78.2, 76.4, 75.5, 72.3, 71.7,67.8, 67.7, 67.4, 64.5, 64.0, 25.7, 21.1, 18.6, 18.6, 15.0 ppm;elemental analysis calcd (%) for C₃₀H₃₈N₆O₇S: C, 57.5, H, 6.1, N, 13.4,S, 5.1. found: C, 57.5, H, 6.2, N, 13.2, S, 5.2.

Allyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (15)

Donor 14 (188 mg, 0.3 mmol) and acceptor 9 (54 mg, 0.2 mmol) weredissolved in dry dichloromethane (6 mL) with molecular sieves, then NIS(72 mg, 0.32 mmol) and trifluoromethanesulfonic acid (9 μL, 0.1 mmol)were added at −30° C. The reaction was stirred at this temperature for 5hours and then filtered through celite. The mixture was washed withNa₂S₂O₃ then KHCO₃. Trisaccharide 15 (114 mg, 65%) was obtained pureafter flash chromatography (hexane/ethyl acetate 10:1): [α]_(D) ²⁰=+64(c=1.3 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.2-7.4 (m, 10H; H—Ar),5.05 (dd, ³J_(1,2)=1.8 Hz, ³J_(2,3)=3.5 Hz, 1H; H-2^(I)), 4.97 (d,³J_(1,2)=1.9 Hz, 1H; H-1^(II)), 4.9 (d, 1H; H-1^(III)), 4.76 (d, 1H;H-1^(I)), 4.01 (dd, ³J_(2,3)=3.0 Hz, 1H; H-2^(II)), 2.10, 2.09 (2s, 6H;OAc), 1.33 (d, ³J_(5,6)=6.0 Hz, 3H; H-6^(I)), 1.32 (d, ³J_(5,6)=6.6 Hz,3H; H-6^(III)), 1.26 ppm (d, 3H; H-6^(II)); ¹³C NMR (126 MHz, CDCl₃): δ170.0, 169.8, 137.5, 137.1, 133.2, 127-129, 117.9, 101.0, 99.4, 96.2,77.2, 77.1, 75.4, 73.3, 71.8, 71.6, 70.9, 68.5, 68.2, 67.8, 67.1, 66.9,64.5, 63.8, 63.7, 21.0, 20.9, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd forC₃₉H₄₉N₉NaO₁₂ [M+Na]⁺: 858.33929. found: 858.33904.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranose (16)

Allyl glycoside 15 (76 mg, 91 μmol) was dissolved in a solution of AcONain AcOH/H₂O 9:1 (0.2 M, 2 mL) and PdCl₂ (32 mg, 180 μmol) was added. Themixture was stirred overnight at room temperature and neutralized withNaHCO₃. The product was extracted with dichloromethane and washed withwater. Chromatography on silica gel (hexane/ethyl acetate 6:1) gavehemiacetal 16 (54 mg, 73%): ¹H NMR (600 MHz, CDCl₃): δ 7.2-7.4 (m, 10H;H—Ar), 5.4 (dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(III)), 5.14(br s, 1H; H-1^(I)), 5.08 (dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.3 Hz, 1H;H-2^(I)), 4.98 (d, ³J_(1,2)=2.0 Hz, 1H; H-1^(II)), 4.9 (d, 1H;H-1^(III)), 2.11, 2.09 (2s, 6H; OAc), 1.33 (d, 3H; H—O, 1.32 (d, 3H;H-6^(III)), 1.26 ppm (d, 3H; H-6^(II)); ¹³C NMR (126 MHz, CDCl₃): δ170.1, 169.8, 137.5, 137.1, 127-129, 101.0, 99.4, 91.8, 77.2, 76.6,75.4, 73.4, 71.8, 71.6, 71.2, 68.2, 67.8, 67.2, 66.9, 64.5, 63.8, 63.7,21.0, 20.9, 18.5, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd forC₃₆H₄₅N₉NaO₁₂ [M+Na]⁺: 818.30799. found: 818.30635.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosylN-phenyltrifluoroacetimidate (17)

Compound 17 (147 mg, 79%) was prepared from trisaccharide 16 (153 mg,192 μmol) as described for 11 and obtained as a mixture α/β 3:2: ¹H NMR(600 MHz, CDCl₃): δ(α) 7.4-6.8 (m, 15H; H—Ar), 6.12 (br s, 1H; H-1^(I)),5.63 (br s, 1H; H-2^(I)), 5.39 (dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.1 Hz,1H; H-2^(III)), 4.97 (br s, 1H; H-1^(II)), 4.89 (d, 1H; H-1^(III)), 4.0(br s, 1H; H-2^(II)), 2.16, 2.08 (2s, 6H; OAc), 1.46 (d, ³J_(5,6)=6.2Hz, 3H; H-6^(I)), 1.29 (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(III)), 1.26 ppm (d,³J_(5,6)=6.2 Hz, 3H; H-6^(II)); HRMS (ESI): m/z calcd forC₄₄H₄₉F₃N₁₀NaO₁₂ [M+Na]⁺: 989.33757. found: 989.33761.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranose (18)

To a solution of disaccharide thioglycoside 14 (210 mg, 0.33 mmol) inacetone (6 mL), NIS (90 mg, 0.44 mmol) and water (100 μL) were added at0° C. and stirred at this temperature for 30 min. and then overnight atroom temperature. After completion, NaHCO₃ solid was added and acetoneevaporated. An extraction dichloromethane/water and then a purificationon silica gel gave the hemiacetal 18 (144 mg, 76%): ¹H NMR (600 MHz,CDCl₃): δ(α) 7.4-7.2 (m, 10H; H—Ar), 5.42 (dd, ³J_(1,2)=2 Hz,³J_(2,3)=3.2 Hz, 1H; H-2^(II)), 5.12 (dd, ³J_(1,2)=1.8 Hz, ³J_(1,OH)=3.5Hz, 1H; H-1^(I)), 4.87 (d, 1H; H-1^(II)), 3.89 (dd, ³J_(2,3)=3 Hz, 1H;H-2^(I)), 2.10 (s, 3H; Ac), 1.30 ppm (2d, 6H; H-6^(I), H-6^(II)); ¹³CNMR (126 MHz, CDCl₃): δ(α) 169.9, 137.7, 137.2, 129-127, 99.5, 93.6,77.3, 75.5, 74.1, 72.3, 71.7, 67.7, 67.4, 67.3, 64.3, 64.0, 21.1, 18.8,18.7 ppm; elemental analysis calcd (%) for C₂₈H₃₄N₆O₈: C, 57.7, H, 5.9,N, 14.4. found: C, 57.9, H, 6.0, N, 14.0.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosylN-phenyltrifluoroacetimidate (19)

Disaccharide 18 (538 mg, 0.92 mmol) was dissolved in dry dichloromethane(9 mL), N-phenyl trifluoroacetimidoyl chloride (350 μL, 2.8 mmol) andCs₂CO₃ (0.9 g, 2.8 mmol) were added and the mixture was stirredovernight at room temperature. After evaporation of the solventchromatography on silica gel (toluene/ethyl acetate 1:0 to 10:2) gavepure imidate 19 (623 mg, 90%): ¹H NMR (500 MHz, CDCl₃): δ(α) 7.4-6.7 (m,15H; H—Ar), 6.05 (br s, 1H; H-1^(I)), 5.41 (br s, 1H; H-2^(II)), 4.86(br s, 1H; H-1^(II)), 3.85 (br s, 1H; H-2^(I)), 2.10 (s, 3H; Ac), 1.33(d, ³J_(5,6)=6.2 Hz, 3H; H-6^(I)), 1.18 ppm (d, ³J_(5,6)=6.2 Hz, 3H;H-6^(II)); ¹⁹F NMR (469 MHz, CDCl₃): δ(α) −75.7 ppm; ¹³C NMR (125 MHz,CDCl₃): δ 170.0, 143.3, 137.3, 137.2, 129-128, 124.7, 119.5, 99.5, 95.7,77.1, 75.5, 72.9, 72.2, 71.8, 70.3, 68.0, 67.2, 63.9, 63.5, 21.1, 18.7,18.5 ppm; HRMS (ESI): m/z calcd for C₃₆H₃₈F₃KN₇O₈ [M+K]⁺: 792.2366.found: 792.2363.

5-Methoxycarbonylpentyl2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (21)

Donor 19 (110 mg, 0.15 mmol) and 5-methoxycarbonylpentanol 20 (ElFangour et al. (2004) J. Org. Chem. 69, 2498-2503) (30 mg, 0.2 mmol) insolution in toluene (7 mL) with some molecular sieves were heated at100° C. and TMSOTf (2 μL, 10 μmol) was added. After heating 1 hour atthis same temperature, the reaction was quenched with pyridine, and themixture filtered. Compound 21 (59 mg, 53%) was obtained afterpurification on silica gel chromatography column (eluent: ethylacetate/hexane 1/6): [α]_(D) ²⁰=+78 (c=1.0 in CHCl₃); ¹H NMR (500 MHz,CDCl₃): δ 7.2-7.4 (m, 10H; H—Ar), 5.41 (dd, 1H, ³J_(1,2)=1.9 Hz,³J_(2,3)=3.2 Hz, H-2^(II)), 4.86 (d, 1H, ³J_(1,2)=1.8 Hz, H-1^(II)),4.66 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(I)), 3.84 (dd, ³J_(1,2)=2 Hz,³J_(2,3)=2.8 Hz, 1H; H-2^(I)), 2.33 (t, ³J_(d,e)=7.5 Hz, 2H; H-e), 2.09(s, 3H; Ac), 1.65 (quint, ³J_(c,d)=³J_(d,e)=7.6 Hz, 2H; H-d), 1.57 (m,2H; H-b), 1.36 (m, 2H; H-c), 1.31 (d, ³J_(5,6)=6 Hz, 3H; H-6^(II)), 1.29ppm (d, ³J_(5,6)=6 Hz, 3H; H-6^(I)); ¹³C NMR (125 MHz, CDCl₃): δ 174.1,169.9, 137.7, 137.2, 129-128, 99.5, 98.7, 77.9, 75.5, 74.1, 72.2, 71.7,67.7, 67.7, 67.4, 67.2, 64.3, 64.0, 51.6, 34.1, 29.2, 25.8, 24.8, 21.1,18.7, 18.6 ppm; elemental analysis calcd (%) for C₃₅H₄₆N₆O₁₀: C, 59.1,H, 6.5, N, 11.8. found: C, 59.0, H, 6.6, N, 11.4.

5-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (22)

Compound 21 (70 mg, 98 μmol) in methanol (3 mL) was treated with a 0.1 Msolution of sodium methoxide (0.1 mL). After 1 h, the reaction wascomplete and neutralized with ion exchange resin H⁺. Filtration andremoval of the solvent under vacuum gave quantitatively pure acceptor 22(65 mg): [α]_(D) ²⁰=+90 (c=1.7 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ7.3-7.4 (m, 10H; H—Ar), 4.94 (d, ³J_(1,2)=1.7 Hz, 1H; H-1^(II)), 4.67(d, ³J_(1,2)=2.1 Hz, 1H; H-1^(I)) 3.99 (dd, ³J_(2,3)=3.1 Hz, 1H;H-2^(II)), 3.84 (dd, ³J_(2,3)=3.0 Hz, 1H; H-2^(I)), 3.35 (dt, ²J=9.7 Hz,³J_(a,b)=6.4 Hz, 1H; H-a), 2.33 (t, ³J_(d,e)=7.5 Hz, 2H; H-e), 1.65(quint, ³J_(c,d)=7.6 Hz, 2H; H-d), 1.57 (m, 2H; H-b), 1.36 (m, 2H; H-c),1.30 (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(II)), 1.30 ppm (d, 3H; H-6^(I)); ¹³CNMR (125 MHz, CDCl₃): δ 174.1, 137.6, 137.3, 129-128, 101.0, 98.9, 78.0,77.8, 74.1, 72.3, 72.2, 67.7, 67.4, 67.3, 67.2, 64.5, 64.0, 51.6, 34.1,29.2, 25.8, 24.8, 18.7, 18.6 ppm; elemental analysis calcd (%) forC₃₃H₄₄N₆O₉: C, 59.3, H, 6.6, N, 12.6. found: C, 59.2, H, 6.45, N, 12.2.

5-Methoxycarbonylpentyl2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (23)

Trisaccharide imidate 17 (45 mg, 47 μmol) and disaccharide alcohol 22(17 mg, 25 μmol) were dissolved in dichloromethane (1 mL) and TMSOTf (1μL, 5 μmol) was added at 0° C. The mixture was stirred 30 min. at 0° C.and 1 hour at room temperature before being quenched with one drop ofpyridine. Purification on silica gel column (eluent: ethylacetate/hexane 1/10) gave pure pentasaccharide 23 (25 mg, 68%): [α]_(D)²⁰=+80 (c=1.0 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.2-7.4 (m, 20H;H—Ar), 5.41 (dd, ³J_(1,2)=1.7 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(V)), 5.13(dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(III)), 5.00 (d,³J_(1,2)=1.7 Hz, 1H; H-1^(II)), 4.95 (d, ³J_(1,2)=1.5 Hz, 1H; H-1^(IV)),4.90 (d, 1H; H-1^(V)), 4.82 (d, 1H; H-1^(III)), 4.61 (br s, 1H;H-1^(I)), 3.98 (dd, ³J_(2,3)=2.9 Hz, 1H; H-2^(II)), 3.84 (dd,³J_(2,3)=2.3 Hz, 1H; H-2^(IV)), 3.81 (dd, ³J_(2,3)=2.3 Hz, 1H; H-2^(I)),3.68 (s, 3H; H-g), 3.58 (dt, ²J=9.7 Hz, ³J_(a,b)=6.6 Hz, 1H; H-a), 3.34(dt, ³J_(a,b)=6.4 Hz, 1H; H-a), 2.32 (t, ³J_(d,e)=7.5 Hz, 2H; H-e), 2.1(s, 6H; Ac), 1.65 (quint, ³J_(c,d)=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b),1.34 (m, 2H; H-c), 1.30 (d, 3H; H-6^(V)), 1.29 (d, 3H; H-6^(IV)), 1.28(d, 3H; H-6^(I)), 1.25 (d, 3H; H-6^(II)), 1.18 ppm (d, 3H; H-6^(III));¹³C NMR (125 MHz, CDCl₃): δ 174.1, 169.9, 169.6, 137.7, 137.5, 137.5,137.2, 129-128, 100.8, 100.3, 99.6, 98.8, 98.8, 77.8, 77.4, 76.8, 76.1,75.5, 74.3, 73.9, 73.7, 72.3, 72.2, 72.0, 71.8, 70.6, 67.6, 68.4, 67.9,67.9, 67.8, 67.3, 67.2, 64.6, 64.6, 64.2, 64.0, 63.9, 51.7, 34.1, 29.2,25.8, 24.8, 21.1, 21.0, 18.8, 18.7, 18.6, 18.5, 18.4 ppm; HRMS (ESI):m/z calcd for C₆₉H₈₇N₁₅NaO₂₀ [M+Na]⁺: 1468.6144. found: 1468.6140.

5-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-Dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (24)

Pentasaccharide 23 (82 mg, 57 μmol) in methanol (3 mL) was treated witha 0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred1 hour at room temperature and then neutralized with ion exchange resinH⁺. Filtration, evaporation and column chromatography (eluent: ethylacetate/toluene 1:6) gave pure compound 24 (67 mg, 86%): [α]_(D) ²⁰=+80(c=1.1 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.2-7.4 (m, 20H; H—Ar),5.04 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(IV)), 4.98 (d, ³J_(1,2)=1.7 Hz, 1H;H-1^(II)), 4.95 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(V)), 4.86 (d, ³J_(1,2)=1.7Hz, 1H; H-1^(III)), 4.62 (br s, 1H; H-1^(I)), 4.06 (dd, ³J_(2,3)=2.9 Hz,1H; H-2^(IV)), 3.99 (dd, ³J_(2,3)=3.1 Hz, 1H; H-2^(II)), 3.94 (dd,³J_(2,3)=2.9 Hz, 1H; H-2^(V)), 3.93 (dd, ³J_(2,3)=3.1 Hz, 1H;H-2^(III)), 3.83 (app. t, ³J_(1,2)=³J_(2,3)=2.9 Hz, 1H; H-2^(I)), 3.68(s, 3H; H-g), 3.34 (dt, ³J_(a,b)=6.4 Hz, 1H; H-a), 3.24 (t, 1H;H-4^(I)), 2.32 (t, ³J_(d,e)=7.5 Hz, 2H; H-e), 1.65 (quint, ³J_(c,d)=7.6Hz, 2H; H-d), 1.56 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.32 (d, 3H;H-6^(II)), 1.29 (d, 6H; H-6^(IV), H-6^(V)), 1.29 (d, 3H; H-6^(I)), 1.17ppm (d, 3H; H-6^(III)); ¹³C NMR (125 MHz, CDCl₃): δ 174.1, 137.5, 137.5,137.4, 137.3, 129-128, 101.1, 100.8, 100.7, 100.5, 98.8, 79.0, 77.9,77.8, 77.8, 77.3, 73.8, 73.5, 73.4, 72.4, 73.4, 72.3, 72.3, 69.6, 67.7,68.4, 67.9, 67.6, 67.6, 67.3, 67.3, 64.6, 64.4, 64.0, 63.9, 63.9, 51.7,34.1, 29.2, 25.8, 24.8, 18.7, 18.7, 18.7, 18.5, 18.4 ppm; HRMS (ESI):m/z calcd for C₆₅H₈₃N₁₅NaO₁₈ [M+Na]⁺: 1384.5933. found: 1384.5926.

5-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (1)

A solution of pentasaccharide 24 (67 mg, 49 μmol) in a mixturepyridine/NEt₃ 1:1 (6 mL) was saturated with H₂S for 1 h and the mediawas then stirred for 24 h at room temperature. The solvent wasco-evaporated with toluene and mass spectrometry of the crude productshowed only one peak corresponding to compound 25 and no productsarising from incomplete reduction: HRMS (ESI): m/z calcd for C₆₅H₉₄N₅O₁₈[M+H]⁺: 1232.6588. found: 1232.6577. This crude material was directlyused for formylation.

Compound 25 was dissolved in methanol (5 mL) and a solution of aceticanhydride/formic acid 2:1 (0.5 mL) was added. The mixture was stirred atroom temperature overnight. The solvent was evaporated andpentasaccharide 26 (43 mg, 63% over 2 steps) was purified with achromatography column (eluent: MeOH/DCM 1:10): HRMS (ESI): m/z calcd forC₇₀H₉₃N₅NaO₂₃ [M+Na]⁺: 1394.6154. found: 1394.6151.

Compound 26 (43 mg, 31 μmol) in solution in acetic acid (8 mL) withpalladium on charcoal was stirred overnight at room temperature underatmosphere of hydrogen. After filtration and concentration,pentasaccharide 1 (17 mg, 54%) was purified on a reverse phase HPLCcolumn (MeCN/H₂O 15:85): ¹H NMR (600 MHz, D₂O): δ 8.21-8.19 (Z) and8.02-8.00 (E) (m, 5H; NCHO), 5.20-4.90 (m, 5H; 5×H-1), 3.72-3.66 (m, 4H,H-a, H-g), 3.56-3.50 (m, 1H; H-a), 2.40 (t, ³J_(d,e)=7.4 Hz, 2H; H-e),1.65-1.56 (m, 4H; H-b, H-d), 1.41-1.32 (m, 2H; H-c), 1.30-1.18 ppm (m,15H; 5×H-6); HRMS (ESI): m/z calcd for C₄₂H₆₉N₅NaO₂₃ [M+Na]⁺: 1034.4276.found: 1034.4275.

Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (27)

Disaccharide 14 (570 mg, 0.91 mmol) in methanol (10 mL) was treated witha 0.1 M solution of sodium methoxide (0.3 mL). The reaction was stirred2 hours at room temperature and then neutralized with ion exchange resinH⁺. Filtration, evaporation and column chromatography (eluent: ethylacetate/hexane 1:4) gave pure alcohol 27 (433 mg, 81%): [α]_(D) ²⁰=+175(c=1.0 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.5-7.3 (m, 10H; H—Ar),5.18 (d, ³J_(1,2)=1.4 Hz, 1H; H-1^(I)), 4.91 (d, ³J_(1,2)=1.5 Hz, 1H;H-1^(II)), 3.99 (ddd, ³J_(2,3)=3.2 Hz, ³J_(2,OH)=1.6 Hz, 1H; H-2^(II)),3.96 (dd, ³J_(2,3)=3 Hz, 1H; H-2^(I)), 2.61 (dq, ³J=7.4 Hz, ²J=12.9 Hz,1H; S—CH₂), 2.55 (dq, ³J=7.5 Hz, 1H; S—CH₂), 1.30 (d, 6H; H-6^(I),H-6^(II)), 1.27 ppm (t, 3H; S—CH₂—CH₃); ¹³C NMR (126 MHz, CDCl₃): δ137.4, 137.2, 129-128, 101.1, 83.6, 78.3, 77.8, 76.1, 72.3, 72.3, 67.7,67.5, 67.3, 64.7, 64.0, 25.8, 18.6, 18.6, 15.0 ppm; HRMS (ESI): m/zcalcd for C₂₈H₃₆N₆NaO₆S [M+Na]⁺: 607.2309. found: 607.2303.

Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (28)

Donor 19 (226 mg, 0.30 mmol) and acceptor 27 (100 mg, 0.17 mmol) weredissolved in toluene (3 mL) with molecular sieves and TMSOTf (2 μL, 11μmol) was added at 100° C. The mixture was stirred 1 hour at 100° C.before being quenched with one drop of pyridine. After filtration, apurification on silica gel column using toluene to elute the leavinggroup of the donor and then a mixture ethyl acetate/toluene 5:95 gavepure tetrasaccharide 28 (159 mg, 81%): [α]_(D) ²⁰=+122 (c=1.0 in CHCl₃);¹H NMR (600 MHz, CDCl₃): δ 7.4-7.3 (m, 20H; H—Ar), 5.41 (dd,³J_(1,2)=1.8 Hz, ³J_(2,3)=3.4 Hz, 1H; H-2^(IV)), 5.10 (d, ³J_(1,2)=1.4Hz, 1H; H-1^(I)), 4.95 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(III)), 4.85 (d, 1H;H-1^(IV)), 4.84 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(II)), 3.88 (dd,³J_(2,3)=2.9 Hz, 1H; H-2^(III)), 3.85 (dd, ³J_(2,3)=2.9 Hz, 1H;H-2^(I)), 3.83 (dd, ³J_(2,3)=2.9 Hz, 1H; H-2^(II)), 2.59 (dq, ³J=7.4 Hz,²J=13 Hz, 1H; S—CH₂), 2.53 (dq, ³J=7.5 Hz, 1H; S—CH₂), 2.11 (s, 3H; Ac),1.27 (2d, 6H; H-6^(I), H-6^(II)), 1.25 (t, 3H; S—CH₂—CH₃), 1.21 (d, 3H;H-6^(IV)), 1.20 ppm (d, 3H; H-6^(III)); ¹³C NMR (126 MHz, CDCl₃): δ169.9, 137.6, 137.3, 137.3, 137.2, 129-128, 100.7, 100.2, 99.3, 83.577.9, 77.0, 76.8, 76.2, 75.6, 73.6, 73.6, 72.4, 72.3, 72.2, 71.7(CH₂-Ph), 68.1, 68.0, 67.8, 67.7, 67.3, 64.7, 64.4, 64.2, 64.0, 25.7,21.1, 18.8, 18.7, 18.6, 18.5, 15.0 ppm; HRMS (ESI): m/z calcd forC₅₆H₆₈N₁₂NaO₁₃S [M+Na]⁺: 1171.4642. found: 1171.4644.

Allyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (29)

Donor 28 (197 mg, 171 μmol) and acceptor 9 (37 mg, 136 μmol) weredissolved in dry dichloromethane (5 mL) with molecular sieves, then NIS(46 mg, 200 μmol) and trifluoromethanesulfonic acid (6 μL, 68 μmol) wereadded at 0° C. The reaction was stirred at this temperature for 15 min.and then filtered through celite. The mixture was washed with Na₂S₂O₃then KHCO₃. Pentasaccharide 29 (125 mg, 68%) was obtained pure afterflash chromatography (hexane/ethyl acetate 6:1): [α]_(D) ²⁰=+88 (c=1.0in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.5-7.2 (m, 20H; H—Ar), 5.41 (dd,³J_(1,2)=1.8 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(V)), 5.41 (dd, ³J_(1,2)=1.8Hz, ³J_(2,3)=3.3 Hz, 1H; H-21), 4.93 (m, 3H; H-1^(II), H-1^(III),H-1^(IV)), 4.85 (d, 1H; H-1^(V)), 4.75 (d, 1H; H-1^(I)), 3.87 (dd,³J_(1,2)=1.8 Hz, ³J_(2,3)=2.9 Hz, 1H; H-2^(IV)), 3.82 (dd, ³J_(1,2)=1.8Hz, ³J_(2,3)=2.9 Hz, 1H; H-2^(III)), ³2.10, 2.08 (2s, 6H; Ac), 1.33 (d,3H; H-6^(I)), 1.29 (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(III)), 1.24 (d,³J_(5,6)=6.2 Hz, 3H; H-6^(II)), 1.20 (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(V)),1.14 ppm (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(IV)); ¹³C NMR (126 MHz, CDCl₃): δ170.1, 169.9, 137.6, 137.4, 137.3, 137.3, 129-128, 133.3 118.1, 101.2,100.5, 100.3, 99.2, 96.4, 77.4, 77.0, 76.8, 75.6, 73.6, 73.5, 73.3,72.4, 72.2, 72.0, 71.7, 71.0, 68.6, 68.3, 68.2, 68.0, 67.8, 67.3, 67.1,64.6, 64.4, 64.2, 64.1, 64.0, 21.1, 21.0, 18.7, 18.6, 18.6, 18.6, 18.5ppm; HRMS (ESI): m/z calcd for C₆₅H₇₉N₁₅NaO₁₈ [M+Na]⁺: 1380.562. found:1380.5611.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranose (30)

Allyl glycoside 29 (40 mg, 29 μmol) was dissolved in a solution ofAcOH/H₂O 9:1 (0.6 mL), AcONa (10 mg, 122 μmol) and PdCl₂ (10 mg, 56μmol) were added. The mixture was stirred overnight at room temperatureand neutralized with NaHCO₃. The product was extracted withdichloromethane, washed with water, dried over MgSO₄, filtrated andconcentrated. Chromatography on silica gel (hexane/ethyl acetate 2:1)gave compound 30 (24 mg, 62%): ¹H NMR (600 MHz, CDCl₃): δ 7.4-7.25 (m,20H; H—Ar), 5.40 (dd, ³J_(1,2)=1.8 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(V)),5.13 (br s, 1H; H-1^(I)), 5.06 (dd, ³J_(1,2)=1.8 Hz, ³J_(2,3)=3.3 Hz,1H; H-2^(I)), 4.94 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(II)), 4.92 (d,³J_(1,2)=1.7 Hz, 2H; H-1^(III), H-1^(IV)), 4.84 (d, 1H; H-1^(V)), 3.98(dd, ³J_(2,3)=2.8 Hz, 1H; H-2^(II)), 3.87 (dd, ³J_(2,3)=2.9 Hz, 1H;H-2^(IV)), 3.82 (dd, ³J_(2,3)=2.8 Hz, 1H; H-2^(III)), 2.10, 2.08 (2s,6H; Ac), 1.33 (d, 3H; H-6^(I)), 1.29 (d, ³J_(5,6)=6.2 Hz, 3H;H-6^(III)), 1.24 (d, ³J_(5,6)=6.2 Hz, 3H; H-6^(II)), 1.20 (d,³J_(5,6)=6.2 Hz, 3H; H-6^(V)), 1.14 ppm (d, ³J_(5,6)=6.2 Hz, 3H;H-6^(IV)). ¹³C NMR (126 MHz, CDCl₃): δ 170.2, 170.0, 137.6, 137.4,137.3, 137.3, 129-128, 101.2, 100.5, 100.3, 99.2, 92.0, 77.0, 76.8,76.6, 76.6, 75.6, 73.6, 73.6, 73.4, 72.4, 72.2, 72.0, 71.7, 71.2, 68.3,68.2, 68.0, 67.8, 67.3, 67.1, 64.6, 64.4, 64.2, 64.1, 64.0, 21.1, 21.0,18.7, 18.6, 18.6, 18.6, 18.5 ppm. HRMS (ESI): m/z calcd forC₆₂H₇₅N₁₅NaO₁₈ [M+Na]⁺: 1340.5307. found: 1340.5291.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosylN-phenyltrifluoroacetimidate (31)

To a solution of free pentasaccharide 30 (118 mg, 89 μmol) indichloromethane (2 mL) were added N-phenyl trifluoroacetimidoyl chloride(34 μL, 270 μmol) and Cs₂CO₃ (90 mg, 280 μmol). The mixture was stirredovernight at room temperature. After filtration, a purification onsilica gel column (eluent: ethyl acetate/hexane 1:9) gave donor 31 (104mg, 79%) in a α/β mixture which was used directly for the nextglycosylation: HRMS (ESI): m/z calcd for C₇₀H₇₉F₃N₁₆NaO₁₈ [M+Na]⁺:1511.5603; found: 1511.5600.

5-Methoxycarbonylpentyl2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (32)

Donor 19 (117 mg, 160 μmol) and acceptor 22 (65 mg, 97 μmol) weredissolved in toluene (2 mL) with molecular sieves and TMSOTf (2 μL, 11μmol) was added at 100° C. The mixture was stirred 1 hour at 100° C.before being quenched with one drop of pyridine. After filtration, apurification on silica gel column using toluene to elute the leavinggroup of the donor and then a mixture ethyl acetate/toluene 5:95 gavepure tetrasaccharide 32 (93 mg, 77%): [α]_(D) ²⁰=+55 (c=1.0 in CHCl₃);¹H NMR (600 MHz, CDCl₃): δ 7.4-7.3 (m, 20H; H—Ar), 5.40 (dd,³J_(1,2)=1.8 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(IV)), 4.94 (d, ³J_(1,2)=1.8Hz, 1H; H-1^(III)), 4.87 (d, ³J_(1,2)=1.9 Hz, 1H; H-1^(II)), 4.84 (d,1H; H-1^(IV)), 4.60 (d, ³J_(1,2)=1.5 Hz, 1H; H-1^(I)), 3.87 (dd,³J_(2,3)=3 Hz, 1H; H-2^(III)), 3.83 (dd, ³J_(2,3)=2.9 Hz, 1H; H-2^(II)),3.78 (dd, ³J_(2,3)=2.9 Hz, 1H; H-2^(I)), 3.68 (s, 3H; H-g), 3.57 (dt,³J_(a,b)=6.6 Hz, ²J=9.6 Hz, 1H; H-a), 3.32 (dt, ³J_(a,b)=6.4 Hz, 1H;H-a), 2.32 (t, ³J_(d,e)=7.5 Hz, 2H; H-e), 2.11 (s, 3H; Ac), 1.64 (quint,³J_(c,d)=7.5 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.26(2d, 6H; H-6^(I), H-6^(II)), 1.20 (d, 3H; H-6^(IV)), 1.16 ppm (d, 3H;H-6^(III)); ¹³C NMR (151 MHz, CDCl₃): δ 174.1, 169.9, 137.6, 137.5,137.3, 137.3, 129-128, 100.6, 100.2, 99.2, 98.7, 77.6, 77.0, 76.8, 75.6,74.2, 73.6, 73.6, 72.4, 72.3, 72.2, 71.7, 68.0, 67.9, 67.8, 67.7, 67.3,67.2, 64.5, 64.4, 64.2, 64.0, 51.7, 34.1, 29.2, 25.8, 24.8, 21.1, 18.8,18.7, 18.6, 18.5 ppm; elemental analysis calcd (%) for C₆₁H₇₆N₁₂O₁₆: C,59.40, H, 6.21, N, 13.63. found: C, 59.62, H, 5.93, N, 13.36.

5-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (33)

Tetrasaccharide 32 (90 mg, 73 μmol) in methanol (3 mL) was treated witha 0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred2 hours at room temperature and then neutralized with ion exchange resinH⁺. Filtration, evaporation and chromatography on silica gel (eluent:ethyl acetate/hexane 1:6) gave pure alcohol 33 (73 mg, 84%): [α]_(D)²⁰=+104 (c=1.0 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.4-7.3 (m, 20H;H—Ar), 4.97 (d, ³J_(1,2)=1.8 Hz, 1H; H-1^(IV)), 4.95 (d, ³J_(1,2=)1.9Hz, 1H; H-1^(III)), 4.87 (³J_(1,2)=1.9 Hz, 1H; H-1^(II)), 4.60 (d,³J_(1,2)=1.8 Hz, 1H; H-1^(I)), 5.40 (dd, ³J_(2,3)=3.0 Hz, 1H; H-2^(IV)),3.87 (dd, ³J_(2,3)=3.0 Hz, 1H; H-2^(III)), 3.83 (dd, ³J_(2,3)=2.9 Hz,1H; H-2^(II)), 3.78 (dd, ³J_(2,3)=2.9 Hz, 1H; H-2^(I)), 3.32 (dt,³J_(a,b)=6.4 Hz, 1H; H-a), 2.32 (t, ³J_(d,e)=7.6 Hz, 2H; H-e), 2.30 (br.s, 1H; OH), 1.64 (quint, ³J_(c,d)=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b),1.34 (m, 2H; H-c), 1.26 (2d, 6H; H-6^(I), H-6^(II)), 1.20 (d, 3H;H-6^(IV)), 1.17 ppm (d, 3H; H-6^(III)); ¹³C NMR (126 MHz, CDCl₃): δ174.1, 137.5, 137.5, 137.3, 137.3, 129-128, 100.6, 100.5, 100.4, 98.7,77.8, 77.6, 77.1, 76.7, 74.1, 73.7, 73.4, 72.4, 72.3, 72.3, 72.2, 67.9,67.9, 67.6, 67.5, 67.3, 67.2, 64.5, 64.4, 64.3, 64.0, 51.7, 34.1, 29.2,25.8, 24.8, 18.8, 18.7, 18.7, 18.5 ppm; HRMS (ESI): m/z calcd forC₅₉H₇₄N₁₂NaO₁₅ [M+Na]⁺: 1213.5289. found: 1213.5278.

5-Methoxycarbonylpentyl2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (34)

Donor 31 (104 mg, 70 μmol) and acceptor 33 (73 mg, 61 μmol) weredissolved in toluene (1.5 mL) with molecular sieves and TMSOTf (1 μL, 5μmol) was added. The mixture was stirred 3 hours at room temperaturebefore being quenched with one drop of pyridine. After filtration, apurification on silica gel column using toluene with a gradient of ethylacetate (from 0% to 10%) gave pure nonasaccharide 34 (45 mg, 30%):[α]_(D) ²⁰=+95 (c=1.0 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.4-7.2 (m,40H; H—Ar), 5.40 (dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.1 Hz, 1H; H-2^(IX)),5.12 (dd, ³J_(1,2)=1.9 Hz, ³J_(2,3)=3.2 Hz, 1H; H-2^(V)), 4.97 (d,³J_(1,2)=1.5 Hz, 2H; H-1), 4.94 (d, ³J_(1,2)=1.8 Hz, 1H; H-1), 4.93 (d,³J_(1,2)=1.8 Hz, 1H; H-1), 4.86 (d, ³J_(1,2)=1.7 Hz, 1H; H-1), 4.85 (d,³J_(1,2)=1.8 Hz, 1H; H-1), 4.84 (d, ³J_(1,2)=1.7 Hz, 1H; H-1^(IX)), 4.82(d, ³J_(1,2)=1.5 Hz, 1H; H-1^(V)), 4.59 (d, ³J_(1,2)=1.1 Hz, 1H;H-1^(I)), 3.95 (dd, ³J_(2,3)=2.8 Hz, 1H; H-2), 3.87 (dd, ³J_(2,3)=3 Hz,1H; H-2), 3.85 (dd, ³J_(2,3)=2.8 Hz, 1H; H-2), 3.84 (dd, ³J_(2,3)=3 Hz,1H; H-2), 3.83 (dd, ³J_(2,3)=3 Hz, 1H; H-2), 3.80 (dd, ³J_(2,3)=3 Hz,1H; H-2), 3.76 (dd, ³J_(2,3)=3 Hz, 1H; H-2^(I)), 3.57 (dt, ²J=9.7 Hz,³J_(a,b)=6.7 Hz, 1H; H-a), 3.32 (dt, ³J_(a,b)=6.4 Hz, 1H; H-a), 2.32 (t,³J_(d,e)=7.5 Hz, 2H; H-e), 2.10, 2.06 (2s, 6H; Ac), 1.64 (quint,³J_(c,d)=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.33 (m, 2H; H-c),1.27-1.12 ppm (9d, ³J_(5,6)=6.2 Hz, 27H; H-6); ¹³C NMR (126 MHz, CDCl₃):δ 174.1 (C-f), 169.9, 169.6, 137.6, 137.5, 137.5, 137.4, 137.4, 137.3,137.3, 137.3, 129-128, 100.8, 100.6, 100.5, 100.3, 100.2, 100.1, 99.2,98.8, 98.7, 77.6, 77.0, 76.8, 76.8, 76.7, 76.6, 76.6, 76.0, 75.6, 74.3,74.2, 73.7, 73.6, 73.6, 73.5, 73.4, 72.4, 72.4, 72.4, 72.3, 72.2, 72.2,72.1, 71.7, 70.6, 68.4, 68.2, 68.0, 68.0, 68.0, 67.9, 67.8, 67.8, 67.3,67.7, 67.2, 64.6, 64.5, 64.4, 64.4, 64.4, 64.2, 64.2, 64.2, 64.0, 51.7,34.1, 29.2, 25.8, 24.8, 21.1, 21.0, 18.8, 18.7, 18.7, 18.7, 18.6, 18.6,18.5, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C₁₂₁H₁₄₇N₂₇NaO₃₂[M+Na]⁺: 2513.0598; found: 2513.0561.

5-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (35)

Nonaccharide 34 (45 mg, 18 μmol) in methanol (2 mL) was treated with a0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred 2hours at room temperature and then neutralized with ion exchange resinH⁺. Filtration, evaporation and chromatography on silica gel (eluent:ethyl acetate/hexane 1:6) gave pure compound 35 (34 mg, 78%): [α]_(D)²⁰=+100 (c=1.0 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.4-7.3 (m, 40H;H—Ar), 4.99 (d, ³J_(1,2)=1.6 Hz, 1H; H-1), 4.97 (d, ³J_(1,2)=1.6 Hz, 1H;H-1), 4.96 (d, ³J_(1,2)=1.6 Hz, 1H; H-1), 4.94 (br s, 2H; H-1), 4.87 (d,³J_(1,2)=1.6 Hz, 1H; H-1), 4.87 (d, ³J_(1,2)=1.6 Hz, 1H; H-1), 4.86 (d,³J_(1,2)=1.7 Hz, 1H; H-1), 4.59 (d, ³J_(1,2)=1.6 Hz, 1H; H-1^(I)), 3.99(m, 2H; H-2), 3.93 (m, 2H; H-2), 3.91 (br s, 1H; H-2), 3.85 (dd,³J_(1,2)=1.6 Hz, ³J_(2,3) 3 Hz, 1H; H-2), 3.83 (dd, ³J_(1,2)=1.6 Hz,³J_(2,3)=3 Hz, 1H; H-2), 3.81 (dd, ³J_(2,3)=3 Hz, 1H; H-2), 3.77 (dd,³J_(2,3)=3 Hz, 1H; H-2^(I)), 3.68 (s, 3H; H-g), 3.57 (dt, ²J=9.7 Hz,³J_(a,b)=6.7 Hz, 1H; H-a), 3.32 (dt, ³J_(a,b)=6.4 Hz, 1H; H-a), 2.32 (t,³J_(d,e)=7.5 Hz, 2H; H-e), 1.64 (quint, ³J_(c,d)=7.6 Hz, 2H; H-d), 1.55(m, 2H; H-b), 1.33 (m, 2H; H-c), 1.27, 1.27, 1.26, 1.25, 1.20, 1.20,1.19, 1.15, 1.15 ppm (9d, ³J_(5,6)=6.2 Hz, 27H; H-6); ¹³C NMR (126 MHz,CDCl₃): δ 174.1, 137.5, 137.5, 137.5, 137.3, 137.3, 137.3, 137.3, 137.3,129-128, 100.8, 100.7, 100.6, 100.6, 100.5, 100.4, 100.3, 100.2, 98.7,79.0, 77.8, 77.6, 77.5, 77.4, 77.0, 76.8, 76.7, 74.2, 73.6, 73.6, 73.5,73.4, 73.4, 73.3, 72.4, 72.4, 72.4, 72.4, 72.4, 72.3, 72.2, 72.2, 69.6,68.5, 68.2, 68.0, 68.0, 68.0, 67.9, 67.6, 67.5, 67.3 (C-5), 67.2 (C-2),67.7 (C-a), 64.5, 64.4, 64.4, 64.4, 64.3, 64.3, 64.1, 64.0, 64.0, 51.7,34.1, 29.2, 25.8, 24.8, 18.8, 18.7, 18.7, 18.7, 18.6, 18.6, 18.6, 18.5,18.4 ppm; HRMS (ESI): m/z calcd for C₁₁₇H₁₄₃N₂₇NaO₃₀ [M+Na]⁺: 2429.0386.found: 2429.0353.

5-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (2)

A solution of nonasaccharide 35 (34 mg, 14 μmol) in a mixturepyridine/NEt₃ 1:1 (4 mL) was saturated with H₂S for 1 h and the mediawas then stirred for 24 h at room temperature. The solvent wasco-evaporated with toluene and mass spectrometry of the crude productshowed only one peak corresponding to compound 36 and no more productfrom incomplete reduction: HRMS (ESI): m/z calcd for C₁₁₇H₁₆₂N₉O₃₀[M+H]⁺: 2173.1439. found: 2173.1422. This crude material was directlyused for formylation.

Compound 36 was dissolved in methanol (3 mL) and a solution of aceticanhydride/formic acid 2:1 (0.3 mL) was added. The mixture was stirred atroom temperature overnight. The solvent was evaporated andnonasaccharide 37 (21 mg, 62% over 2 steps) was purified with achromatography column (eluent: MeOH/DCM 1:10): HRMS (ESI): m/z calcd forC₁₂₆H₁₆₁N₉Na₂O₃₉ [M+2Na]²⁺: 1235.0338. found: 1235.0333.

Compound 37 (21 mg, 8.6 μmol) in solution in acetic acid (5 mL) withpalladium on charcoal was stirred overnight at room temperature underatmosphere of hydrogen. After filtration and concentration,nonasaccharide 2 (7 mg, 48%) was purified on a reverse phase HPLC column(MeCN/H₂O 18:82): ¹H NMR (600 MHz, D₂O): δ 8.21-8.00 (m, 9H; NCHO),5.21-4.84 (m, 9H; 9×H-1), 4.20-3.78 (m, 34H; 9×H-2, 9×H-3, 7×H-4,9×H-5), 3.74-3.68 (m, 4H; H-a, H-g), 3.56-3.3.34 (m, 3H; 2×H-4, H-a),2.40 (t, ³J_(d,e)=7.4 Hz, 2H; H-e), 1.66-1.58 (m, 4H; H-b, H-d),1.42-1.33 (m, 2H; H-c), 1.30-1.15 ppm (m, 27H; 9×H-6); HRMS (ESI): m/zcalcd for C₇₀H₁₁₃N₉O₃₉Na₂ [M+2Na]²⁺: 874.846. found: 874.8467.

Proton labeling of the linker for compounds 38-41 is as follows:

(2-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (38)

A solution of 1 (12 mg, 11.8 μmol) in freshly distilled1,2-diaminoethane (550 μL) was stirred at 50° C. for 48 h thenconcentrated. The residue was dissolved in water, neutralized withacetic acid and purified by reversed phase HPLC on C18 column ingradient water-acetonitrile to give 38 as a white powder (10 mg, 81%):¹H NMR (500 MHz, D₂O): δ 8.21-8.19 and 8.02-8.00 (m, 5H; NCHO),5.17-4.86 (m, 5H; 5×H-1), 4.19-3.76 (m, 19H; 5×H-2, 5×H-3, 4×H-4,5×H-5), 3.71-3.66 (m, 1H; H-a), 3.55-3.51 (m, 1H; H-a), 3.50-3.32 (m,1H; H-4), 3.27 (t, ³J_(f,g)=6.2 Hz, 2H; H-f), 2.80 (t, 2H; H-g), 2.26(t, ³J_(d,e)=7.4 Hz, 2H; H-e), 1.64-1.54 (m, 4H; H-b, H-d), 1.40-1.31ppm (m, 2H; H-c), 1.30-1.16 (m, 15H; 5×H-6); HRMS (ESI): m/z calcd forC₄₃H₇₄N₇O₂₂ [M+H]⁺: 1040.4881. found: 1040.4879.

1-[(2-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(40)

To a solution of amine 38 (5 mg, 4.8 μmol) in a mixture of water (300μL) and ethanol (200 μL) a solution of3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 26 μL) was addedand pH was adjusted to 8 by addition of sodium bicarbonate solution.After 0.5 h, when TLC indicated the reaction was complete, the reactionmixture was neutralized with acetic acid and purified by reversed phaseHPLC (C18) using gradient water-acetonitrile. Product which came out at20% of acetonitrile was lyophilized to afford squarate 40 as a whitepowder (4.2 mg, 73%): ¹H NMR (600 MHz, D₂O): δ 8.23-8.18 and 8.06-7.98(m, 5H; NCHO), 5.18-5.87 (m, 5H; 5×H-1), 4.69 (m, 2H; H-h), 3.72-3.58(m, 3H; H-a, 2×H-f), 3.51-3.36 (m, 4H; H-4, H-a, 2×H-g). 2.24-2.17 (m,2H; H-e), 1.82-1.74 (m, 2H; H-i), 1.61-1.49 (m, 4H; H-b, H-d), 1.49-1.38(m, 2H; H-j), 1.33-1.18 (m, 17H; 15×H-6, H-c), 0.96-0.91 (m, 3H; H-k);HRMS (ESI): m/z calcd for C₅₁H₈₁N₇NaO₂₅ [M+Na]⁺: 1214.5174. found:1214.5177.

(2-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (39)

Methyl ester 2 (5.9 mg, 3.46 μmol) in freshly distilled ethylenediamine(400 μL) was stirred at 50° C. for 48 h. TLC indicated the reaction wasalmost complete. The mixture was concentrated, dissolved in water andneutralized with 10% acetic acid. It was first purified on a SepPak C18cartridge washing it first with water and then with methanol. Methanolfractions containing the product were combined and concentrated and thenpurified on HPLC (C18) using water-acetonitrile gradient. The productcame out at 12% of acetonitrile. It was concentrated and lyophilized toyield the title amine 39 as a white powder (4.5 mg, 75%): ¹H NMR (500MHz, D₂O): δ 8.22-7.99 (m, 9H; NCHO), 5.20-4.86 (m, 9H; 9×H-1),4.18-3.76 (m, 34H; 9×H-2, 9×H-3, 7×H-4, 9×H-5), 3.72-3.66 (m, 1H; H-a),3.55-3.29 (m, 5H; 2×H-4, H-a, H-f), 2.81 (m, 2H; H-g), 2.26 (t,³J_(d,e)=7.4 Hz, 2H; H-e), 1.65-1.58 (m, 4H; H-b, H-d), 1.40-1.30 (m,2H; H-c), 1.30-1.16 (m, 27H; 9×H-6); HRMS (ESI): m/z calcd forC₇₁H₁₁₈N₁₁O₃₈ [M+H]⁺: 1732.7634. found: 1732.7596.

1-[(2-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(41)

The amine 39 (4.5 mg, 2.5 μmol) was dissolved in water (0.3 mL) andethanol (0.2 mL) was added to the solution. It was stirred at roomtemperature and a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione inethanol (20%, 17 μL) was added and the pH of the reaction mixture wasadjusted to 8 by careful addition of NaHCO₃ solution. When TLC indicatedthe reaction was complete the mixture was neutralized with 10% aceticacid and purified by reversed phase HPLC (C18) using gradientwater-acetonitrile. The product which came out at 20% of acetonitrilewas concentrated and lyophilized to afford title compound 41 as a whitepowder (3.7 mg, 76%): ¹H NMR (500 MHz, D₂O): δ 8.31-8.08 (m, 9H; NCHO),5.30-4.94 (m, 9H; 9×H-1), 4.82-4.74 (m, 2H; H-h), 4.28-3.42 (m, 42H;9×H-2, 9×H-3, 9×H-4, 9×H-5, H-a, H-f, H-g), 2.33-2.25 (m, 2H; H-e),1.91-1.82 (m, 2H; H-i), 1.70-1.57 (m, 4H; H-b, H-d), 1.56-1.48 (m, 2H;H-j), 1.42-1.23 (m, 29H; 9×H-6, H-c), 1.05-0.99 (m, 3H; H-k); HRMS(ESI): m/z calcd for C₇₉H₁₂₅N₁₁Na₂O₄₁[M+2Na]⁺²: 964.8909. found:964.8904.

Methyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranoside (50)(Eichler et al. (1991) Glycoconjugate 8, 69-74)

Benzoyl chloride (2.0 mL, 17.2 mmol) was added dropwise to a stirredsolution of Methyl 4-azido-4,6-dideoxy-α-D-mannopyranoside 3 (Bundle etal. (1998) Carbohydr. Res. 174, 239-251) (1.59 g, 7.82 mmol) in pyridine(5 mL) containing DMAP (0.191 g, 1.56 mmol) at 0° C. The resultingmixture was stirred under argon for 10 h at room temperature. Then CH₃OH(2 mL) was added to the reaction mixture. It was stirred for 10 min,then diluted with CH₂Cl₂ (˜100 mL) and washed with aq. HCl (1M, 2×50mL), water (100 mL), 5% aq. NaHCO₃ (50 mL), and brine (30 mL). Theorganic phase was separated, dried over MgSO₄, and concentrated invacuo. The residue was purified by column chromatography on silica gel(ethyl acetate-hexane gradient elution) to afford the title compound(3.12 g, 97%) as a white foam. Analytical data for 50: Rf=0.40 (ethylacetate/hexane, 1/9, v/v); [α]_(D) ²¹=−130.8 (c=1.1, CHCl₃); ¹H NMR (600MHz, CDCl₃): δ 8.21-7.31 (m, 10H, H—Ar), 5.60 (dd, 1H, J_(2,3)=1.8 Hz,H-2), 5.59 (dd, 1H, J_(3,4)=3.6 Hz, H-3), 4.85 (d, 1H, J_(1,2)=1.2 Hz,H-1), 3.83 (dq, 1H, J_(4,5)=10.2 Hz, J_(5,6)=6.0 Hz, H-5), 3.76 (dd, 1H,J_(4,5)=10.2 Hz, H-4), 3.45 (s, 3H, —OCH₃), 1.48 ppm (d, 3H, J_(5,6)=6.0Hz, H-6); ¹³C NMR (126 MHz, CDCl₃): δ 165.4, 165.3, 133.8, 133.5, 133.3,130.2, 129.8, 129.8, 129.5, 129.3, 128.6, 128.5, 128.4, 98.6, 71.1,69.8, 66.9, 63.5, 55.4, 18.6 ppm; HRMS (ESI): m/z calcd for C₂₁H₂₁N₃O₆Na[M+Na]⁺: 434.1323. found: 434.1317.

1-O-Acetyl-4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranose (51)

A solution of 50 (3.10 g, 7.54 mmol) in acetic anhydride/aceticacid/sulfuric acid (50:20:0.5, 70 mL) was stirred at room temperaturefor 6 h, and then poured into ice-cold 1M K₂CO₃ solution (100 mL). Themixture was then diluted with CH₂Cl₂ (˜200 mL) and washed with water(2×100 mL), 5% aq. NaHCO₃ (50 mL), and brine (30 mL). The organic phasewas separated, dried over MgSO₄, and concentrated in vacuo. The residuewas purified by column chromatography on silica gel (ethyl acetatehexane gradient elution) to afford the title compound (3.04 g, 92%) as awhite foam. Analytical data for 51: Rf=0.30 (ethyl acetate/hexane, 1/9,v/v); [α]_(D) ²¹=−119.8 (c=1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ7.35-8.09 (m, 10H, H—Ar), 6.25 (d, J=1.5 Hz, 1H, H-1), 5.65 (dd,J_(2,3)=3.5 Hz, 1H, H-2), 5.61 (dd, J_(3,4)=10.1 Hz, 1H, H-3), 3.91 (dq,J=10.2, 6.2 Hz, 1H, H-5), 3.85 (dd, J_(4,5)=10.1 Hz, 1H, H-4), 2.24 (s,3H, —OC—CH₃), 1.51 (d, J_(5,6)=6.2 Hz, 3H, H-6); ¹³C NMR (126 MHz,CDCl₃): δ 168.4, 165.4, 165.1, 133.7, 133.5, 129.9, 129.8, 129.1, 129.0,128.6, 128.5, 90.7, 70.9, 69.4, 68.6, 63.0, 21.0, 18.7 ppm; HRMS (ESI):m/z calcd for C₂₂H₂₁N₃O₇Na [M+Na]⁺: 462.1272. found: 462.1265.

4-Azido-2,3-di-O-benzoyl-4,6-dideoxy-α/β-D-mannopyranose (52)

Hydrazine acetate (0.056 g, 0.612 mmol) was added to a stirred solutionof 51 (0.224 g, 0.510 mmol) in DMF (1 mL) under argon atmosphere and thesolution was stirred at 60° C. for 30 min. Then the mixture was cooledto room temperature, diluted with ethyl acetate (50 mL), washed withwater (2×50 mL) and brine (30 mL). The organic phase was separated,dried over MgSO₄, and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (ethyl acetate-hexane gradientelution) to afford the title compound (0.191 g, 95%) as a white foam.Analytical data for 52: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); ¹H NMR(500 MHz, CDCl₃): α:β ratio=7:1; δ 7.11-8.06 (m, 20H, H—Ar), 5.78 (dd,J_(2,3)=3.0 Hz, 1H, H-2_(β)), 5.66 (dd, J_(3,4)=10 Hz, 1H, H-3_(α)),5.63 (dd, J_(2,3)=3.2 Hz, 1H, H-2_(α)), 5.36 (dd, J_(1,2)=1.6 Hz, 1H,H-1_(α)), 5.30 (dd, J_(3,4)=10.3 Hz, 1H, H-3_(β)), 5.07 (dd, J_(1,2)=1.5Hz, 1H, H-1_(β)), 1.52 (d, J_(5,6)=6.0 Hz, 3H, H-6_(β)), 1.45 ppm (d,J_(5,6)=6.2 Hz, 3H, H-6_(α)); ¹³C NMR (126 MHz, CDCl₃): δ 166.1, 165.5,165.4, 133.8, 133.5, 133.4, 130.0, 129.8 (×2), 129.4, 129.2, 129.0 (×2),128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 92.9, 92.1, 73.3, 71.4, 70.9,70.8, 70.2, 67.1, 63.6, 62.8, 18.7, 18.6 ppm; HRMS (ESI): m/z calcd forC₂₀H₁₉N₃O₆Na [M+Na]⁺: 420.1166. found: 420.1163.

4-Azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyltrichloroacetimidate (53)

To a stirred solution of 52 (2.0 g, 5.03 mmol) in CH2Cl2 (20 mL)containing CCl3CN (10.1 mL, 100 mmol), DBU (0.150 mL, 1.00 mmol) wasadded at room temperature under an argon atmosphere. After 10 min,solvents were evaporated in vacuo and the residue was purified by columnchromatography on silica gel (ethyl acetate-hexane gradient elution) toafford the title compound (2.335 g, 86%) as an off-white foam.Analytical data for 53: Rf=0.60 (ethyl acetate/hexane, 1/4, v/v);[α]_(D) ²¹=−101.9 (c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 8.79 (s,1H, N—H), 7.33-8.09 (m, 10H, H—Ar), 6.43 (d, J_(1,2)=1.5 Hz, 1H, H-1),5.85 (dd, J_(2,3)=3.5 Hz, 1H, H-2), 5.68 (dd, J_(3,4)=10.5 Hz, 1H, H-3),4.07 (dq, J=10.2, 6.2 Hz, 1H, H-5), 3.88 (dd, J_(4,5)=10.2 Hz, 1H, H-4),1.53 ppm (d, J_(5,6)=6.2 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃): δ165.3, 165.1, 160.0, 133.7, 133.5, 129.9, 129.8, 129.1, 129.0, 128.7,128.5, 94.7, 90.7, 70.9, 69.9, 68.1, 62.9, 18.7 ppm; HRMS (ESI): m/zcalcd for C₂₂H₁₉Cl₃N₄O₆Na [M+Na]⁺: 563.0262. found: 563.0251.

Methyl 4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside(55)

Benzoyl chloride (0.872 mL, 7.5 mmol) was added dropwise to a stirredsolution of 54 (Peters & Bundle (1989) Can. J. Chem. 67, 497-502) (2.0g, 6.82 mmol) in pyridine (10 mL) containing DMAP (0.166 g, 1.36 mmol)at 0° C. The resulting mixture was stirred under argon for 3 h at roomtemperature. Then CH₃OH (2 mL) was added to the reaction mixture whichwas stirred for 10 min, then diluted with CH₂Cl₂ (˜80 mL) and washedwith aq. HCl (1M, 2×50 mL), water (100 mL), 5% aq. NaHCO₃ (50 mL), andbrine (30 mL). The organic phase was separated, dried over MgSO₄, andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (ethyl acetate-hexane gradient elution) to afford thetitle compound (2.47 g, 91%) as oil. Analytical data for 55: Rf=0.50(ethyl acetate/hexane, 1/9, v/v); [α]_(D) ²¹=−18.8 (c=1.1, CHCl₃); ¹HNMR (600 MHz, CDCl₃): δ 7.13-8.10 (m, 10H, H—Ar), 5.32 (dd, J_(3,4)=10.4Hz, 1H, H-3), 4.71 (d, J_(1,2)=1.4 Hz, 1H, H-1), 3.37 (s, 3H, —OCH₃),1.42 ppm (d, J_(5,6)=6.2 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃): δ165.5, 137.5, 133.3, 129.9, 129.5, 128.5, 128.3, 127.8 (×2), 98.9, 74.8,73.3, 73.2, 67.0, 63.2, 55.0, 18.5 ppm; HRMS (ESI): m/z calcd forC₂₁H₂₃N₃O₅Na [M+Na]⁺: 420.1530. found: 420.1521.

1-O-Acetyl-4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranose(56)

A solution of 55 (1.40 g, 3.52 mmol) in acetic anhydride/aceticacid/sulfuric acid (50:20:0.5, 35 mL) was stirred at room temperaturefor 3 h, and then poured into ice-cold 1M K₂CO₃ solution (100 mL). Themixture was then diluted with CH₂Cl₂ (˜100 mL) and washed with water(2×80 mL), 5% aq. NaHCO₃ (50 mL), and brine (30 mL). The organic phasewas separated, dried over MgSO₄, and concentrated in vacuo. The residuewas purified by column chromatography on silica gel (ethyl acetatehexane gradient elution) to afford the title compound (1.22 g, 81%) as awhite foam. Analytical data for 56: Rf=0.30 (ethyl acetate/hexane, 1/9,v/v); [α]_(D) ²¹=−9.9 (c=1.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ7.10-8.08 (m, 10H, H—Ar), 6.17 (d, J_(1,2)=1.3 Hz, 1H, H-1), 5.29 (dd,J_(3,4)=10.5 Hz, 1H, H-3), 4.00 (dd, J_(2,3)=3.5 Hz, 1H, H-2), 2.15 (s,3H, —OC—CH₃), 1.42 ppm (d, J_(5,6)=6.1 Hz, 3H, H-6); ¹³C NMR (126 MHz,CDCl₃): δ 169.0, 165.6, 136.9, 133.5, 129.9, 129.2, 128.5, 128.4, 128.0,127.9, 91.1, 73.5, 73.0, 72.7, 69.6, 62.6, 21.0, 18.5 ppm; HRMS (ESI):m/z calcd for C₂₂H₂₃N₃O₆Na [M+Na]⁺: 448.1479. found: 448.1471.

4-Azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose (57)

Hydrazine acetate (0.310 g, 3.35 mmol) was added to a stirred solutionof 56 (1.190 g, 2.79 mmol) in DMF (10 mL) under argon atmosphere andstirred at 60° C. for 30 min. Then the mixture was cooled to roomtemperature, diluted with ethyl acetate (100 mL), washed with water(2×80 mL) and brine (30 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-hexane gradient elution) toafford the title compound (0.912 g, 85%) as a white foam. Analyticaldata for 57: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); ¹H NMR (500 MHz,CDCl₃): α:β ratio=5:1; δ 7.16-8.14 (m, 20H, H—Ar), 5.43 (dd,J_(3,4)=10.4 Hz, 1H, H-3_(α)), 5.27 (dd, J_(1,2)=1.8 Hz, J_(1,—OH)=3.3Hz, 1H, H-1_(α)), 5.13 (dd, J_(3,4)=10.5 Hz, 1H, H-3_(β)), 4.82 (dd,J_(1,2)=1.5 Hz, J_(1,—OH)=11.6 Hz, 1H, H-1_(β)), 4.14 (dd, J_(2,3)=3.0Hz, 1H, H-2_(β)), 4.06 (dd, J_(2,3)=3.1 Hz, 1H, H-2_(α)), 1.46 (d,J_(5,6)=6.2 Hz, 3H, H-6_(β)), 1.42 ppm (d, J_(5,6)=6.1 Hz, 3H, H-6_(α));¹³C NMR (126 MHz, CDCl₃): δ 165.6 (×2), 137.4, 137.1, 133.8, 133.4,129.9, 129.5, 128.9, 128.7, 128.5, 128.3 (×2), 128.2, 127.9, 127.8,93.2, 92.7, 76.3, 75.8, 75.7, 75.0, 73.3, 72.9, 70.9, 67.2, 63.2, 62.6,18.6, 18.5 ppm; HRMS (ESI): m/z calcd for C₂₀H₂₁N₃O₅Na [M+Na]⁺:406.1373. found: 406.1366.

4-Azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyltrichloroacetimidate (58)

To a stirred solution of 57 (0.890 g, 2.32 mmol) in CH2Cl2 (15 mL)containing CCl3CN (4.65 mL, 46.4 mmol), DBU (70 μL, 0.464 mmol) wasadded at room temperature under argon atmosphere. After 10 min, solventswere evaporated in vacuo and the residue was purified by columnchromatography on silica gel (ethyl acetate-hexane gradient elution) toafford the title compound (1.136 g, 93%) as an off-white foam.Analytical data for 58: Rf=0.80 (ethyl acetate/toluene, 1/9, v/v);[α]_(D) ²¹=−7.3 (c=1.5, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 8.66 (s, 1H,—C═NH), 7.12-8.10 (m, 10H, H—Ar), 6.36 (d, J_(1,2)=1.8 Hz, 1H, H-1),5.39 (dd, J_(3,4)=7.2 Hz, 1H, H-3), 4.26 (dd, J_(2,3)=3.1 Hz, 1H, H-2),3.91-3.99 (m, 2H, H-4, H-5), 1.48 ppm (d, J_(5,6)=6.0 Hz, 3H, H-6); ¹³CNMR (126 MHz, CDCl₃): δ 165.5, 160.4, 137.0, 133.5, 129.9, 129.2, 128.5,128.4, 128.0 (×2), 127.9, 95.3, 90.8, 73.2, 73.0, 72.8, 70.1, 62.5, 18.6ppm; HRMS (ESI): m/z calcd for C₂₂H₂₁Cl₃N₄O₅Na [M+Na]⁺: 549.0470. found:549.0465.

Methyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside(59)

Benzoyl chloride (0.872 mL, 7.5 mmol) was added dropwise to a stirredsolution of 4 (2.0 g, 6.82 mmol) in pyridine (10 mL) containing DMAP(0.166 g, 1.36 mmol) at 0° C. The resulting mixture was stirred underargon for 3 h at room temperature. Then CH₃OH (2 mL) was added to thereaction mixture, stirred for 10 min, then diluted with CH₂Cl₂ (˜80 mL)and washed with aq. HCl (1M, 2×50 mL), water (100 mL), 5% aq. NaHCO₃ (50mL), and brine (30 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-hexane gradient elution) toafford the title compound (2.32 g, 86%) as a white foam. Analytical datafor 59: Rf=0.50 (ethyl acetate/hexane, 1/9, v/v); [α]_(D) ²¹=−27.9(c=1.7, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.21-8.10 (m, 10H, H—Ar),5.55 (dd, J_(2,3)=3.2 Hz, 1H, H-2), 4.77 (d, J_(1,2)=1.8 Hz, 1H, H-1),3.91 (dd, J_(3,4)=9.7 Hz, 1H, H-3), 3.37 (s, 3H, —OCH₃), 1.38 ppm (d,J_(5,6)=6.0 Hz, 3H, H-6); ¹³C NMR (126 MHz, CDCl₃): δ 165.6, 137.3,133.3, 129.9, 129.7, 128.4, 128.3, 128.1, 127.8, 98.8, 76.1, 71.4, 67.8,66.8, 64.3, 55.1, 18.7 ppm; HRMS (ESI): m/z calcd for C₂₁H₂₃N₃O₅Na[M+Na]⁺: 420.1530. found: 420.1528.

1-O-Acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose(60)

A solution of 59 (2.06 g, 5.19 mmol) in acetic anhydride/aceticacid/sulfuric acid (50:20:0.5, 35 mL) was stirred at room temperaturefor 3 h, and then poured into ice-cold 1M K₂CO₃ solution (100 mL). Themixture was then diluted with CH₂Cl₂ (˜100 mL) and washed with water(2×80 mL), 5% aq. NaHCO₃ (50 mL), and brine (30 mL). The organic phasewas separated, dried over MgSO₄, and concentrated in vacuo. The residuewas purified by column chromatography on silica gel (ethylacetate-hexane gradient elution) to afford the title compound (1.95 g,89%) as a white foam.] α:β ratio=9:1 (isolated yield); Analytical datafor 60: α/β ratio=9:1 (isolated yield); α-anomer: Rf=0.45 (ethylacetate/hexane, 1.5/8.5, v/v); [α]_(D) ²¹=+1.8 (c=1.9, CHCl₃); ¹H NMR(500 MHz, CDCl₃): δ 7.27-8.09 (m, 10H, H—Ar), 6.16 (d, J_(1,2)=2.0 Hz,1H, H-1), 2.13 (s, 3H, —OC—CH₃), 1.40 ppm (d, J_(5,6)=6.0 Hz, 3H, H-6);¹³C NMR (126 MHz, CDCl₃): δ 168.3, 165.3, 136.9, 133.5, 129.9, 129.3,128.5, 128.4, 128.2, 128.0, 91.1, 75.8, 71.6, 69.3, 66.7, 63.8, 20.9,18.7 ppm; HRMS (ESI): m/z calcd for C₂₂H₂₃N₃O₆Na [M+Na]⁺: 448.1479.found: 448.1475; 0-anomer: Rf=0.40 (ethyl acetate/hexane, 1.5/8.5, v/v);[α]_(D) ²¹=−50.2 (c=1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.27-8.13(m, 10H, H—Ar), 5.82 (dd, J_(2,3)=3.5 Hz, 1H, H-2), 5.77 (d, J_(1,2)=1.5Hz, 1H, H-1), 2.03 (s, 3H, —OC—CH₃), 1.46 ppm (d, J_(5,6)=6.0 Hz, 3H,H-6); ¹³C NMR (126 MHz, CDCl₃): δ 168.8, 165.8, 136.6, 133.4, 130.0,129.5, 128.5 (×2), 128.3, 128.1, 91.2, 78.1, 72.1, 71.4, 66.8, 63.6,20.8, 18.6 ppm; HRMS (ESI): m/z calcd for C₂₂H₂₃N₃O₆Na [M+Na]⁺:448.1479. found: 448.1474.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose (61)

Hydrazine acetate (0.572 g, 6.20 mmol) was added to a stirred solutionof 60 (2.20 g, 5.17 mmol) in DMF (7 mL) under argon atmosphere andstirred at 60° C. for 30 min. Then the mixture was cooled to roomtemperature, diluted with ethyl acetate (100 mL), washed with water(2×80 mL) and brine (30 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-hexane gradient elution) toafford the title compound (1.83 g, 93%) as a white foam. Analytical datafor 61: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); ¹H NMR (500 MHz,CDCl₃): α/β ratio=5:1; δ 7.25-8.15 (m, 20H, H—Ar), 5.74 (dd, J_(2,3)=3.5Hz, 1H, H-2_(β)), 5.60 (dd, J_(2,3)=3.1 Hz, 1H, H-2_(α)), 5.32 (d,J_(1,2)=1.3 Hz, 1H, H-1_(α)), 4.89 (s, 1H, H-1_(β)), 1.47 (d,J_(5,6)=6.2 Hz, 3H, H-6_(β)), 1.40 ppm (d, J_(5,6)=6.1 Hz, 3H, H-6_(α));¹³C NMR (126 MHz, CDCl₃): δ 166.3, 165.7, 137.2, 136.8, 133.6, 133.3,130.0, 129.9, 129.6, 129.2, 128.5, 128.4 (×2), 128.3, 128.2, 128.0,127.8, 93.2, 92.5, 78.5, 75.6, 71.5 (×2), 71.3, 69.2, 68.2, 67.1, 64.3,63.7, 18.7 (×2) ppm; HRMS (ESI): m/z calcd for C₂₀H₂₁N₃O₅Na [M+Na]⁺:406.1373. found: 406.1374.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyltrichloroacetimidate (62)

To a stirred solution of 61 (1.820 g, 4.75 mmol) in CH2Cl2 (20 mL)containing CCl3CN (9.50 mL, 95.0 mmol), DBU (140 μL, 0.95 mmol) wasadded at room temperature under argon atmosphere. After 10 min, solventswere evaporated in vacuo and the residue was purified by columnchromatography on silica gel (ethyl acetate hexane gradient elution) toafford the title compound (2.0 g, 80%) as an off-white foam. Analyticaldata for 62: Rf=0.60 (ethyl acetate/hexane, 1/4, v/v); [α]_(D) ²¹=−10.8(c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 8.70 (s, 1H, —C═NH),7.28-8.12 (m, 10H, H—Ar), 6.31 (d, J_(1,2)=1.8 Hz, 1H, H-1), 5.67 (dd,J_(2,3)=3.5 Hz, 1H, H-2), 3.98 (dd, J_(3,4)=10.0 Hz, 1H, H-3), 3.57-3.66(dd, J_(4,5)=10.0 Hz, 1H, H-4), 1.38-1.44 ppm (d, J_(5,6)=6.5 Hz, 3H,H-6); ¹³C NMR (126 MHz, CDCl₃): δ 165.3, 159.8, 136.7, 133.5, 129.9,129.3, 128.6 (×3), 128.5 (×2), 128.4, 128.1, 95.0, 90.7, 75.2, 71.6,69.9, 66.4, 63.7, 18.7 ppm; HRMS (ESI): m/z calcd for C₂₂H₂₁Cl₃N₄O₅Na[M+Na]⁺: 549.0470. found: 549.0475.

Ethyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (63)

A mixture of glycosyl donor 53 (1.010 g, 1.87 mmol), glycosyl acceptor13 Peters & Bundle (1989) Can. J. Chem. 67, 491-496) (0.550 g, 1.70mmol), and freshly activated molecular sieves (3 Å, 2.0 g) in CH₂Cl₂ (30mL) was stirred under argon for 5 h at room temperature. TMSOTf (67 μL,0.374 mmol) was added and the resulting mixture was stirred for anadditional 60 min. Then Et₃N (1 mL) was added, the solid was filteredoff and the residue was rinsed with CH₂Cl₂ (3×20 mL). The combinedfiltrate (˜100 mL) was washed with 20% aq. NaHCO₃ (50 mL), water (50mL), and brine (30 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (1.073 g, 90%) as a white foam. Analyticaldata for 63: Rf=0.70 (ethyl acetate/toluene, 1/9, v/v); [α]_(D) ²¹=−18.4(c=1.1, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.12-8.03 (m, 15H, H—Ar),5.70 (dd, J_(2,3)=3.2 Hz, 1H, H-2^(B)), 5.62 (dd, J_(3,4)=10.3 Hz, 1H,H-3^(B)), 5.24 (d, J_(1,2)=1.5 Hz, 1H, H-1^(A)), 4.98 (d, J_(1,2)=1.5Hz, 1H, H-1^(B)), 2.54-2.67 (m, 2H, S—CH₂—), 1.44 (d, J_(5,6)=6.2 Hz,3H, H-6^(B)), 1.37 (d, J_(5,6)=6.1 Hz, 3H, H-6^(A)), 1.29 ppm (t, J=7.4Hz, 3H, S—CH₂—CH₃); ¹³C NMR (126 MHz, CDCl₃): δ 165.2, 164.9, 137.4,133.4, 133.3, 129.8 (×2), 129.5, 129.3, 128.5 (×2), 128.4, 128.1, 127.9,99.4, 83.3, 78.4, 76.5, 72.4, 70.8, 69.5, 67.8, 67.6, 64.2, 63.5, 25.6,18.6, 18.4, 14.9 ppm; HRMS (ESI): m/z calcd for C₃₅H₃₈N₆O₈SNa [M+Na]⁺:725.2364. found: 725.2350.

Ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl(1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (64)

A mixture of glycosyl donor 62 (1.980 g, 3.76 mmol), glycosyl acceptor13 (1.106 g, 3.42 mmol), and freshly activated molecular sieves (3 Å,4.0 g) in CH₂Cl₂ (30 mL) was stirred under argon for 5 h at roomtemperature. TMSOTf (0.136 mL, 0.753 mmol) was added and the resultingmixture was stirred for additional 1 h. Then Et₃N (1 mL) was added, thesolid was filtered off and the residue was rinsed with CH₂Cl₂ (3×30 mL).The combined filtrate (˜120 mL) was washed with 20% aq. NaHCO₃ (50 mL),water (50 mL), and brine (30 mL). The organic phase was separated, driedover MgSO₄, and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (ethyl acetate-toluene gradientelution) to afford the title compound (2.010 g, 85%) as a white foam.Analytical data for 64: Rf=0.50 (ethyl acetate/hexane, 1/9, v/v);[α]_(D) ²¹=+50.0 (c=1.4, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.21-8.11(m, 15H, H—Ar), 5.63 (dd, J_(2,3)=2.5 Hz, 1H, H-2^(B)), 5.22 (d,J_(1,2)=1.0 Hz, 1H, H-1^(A)), 4.95 (d, J_(1,2)=1.7 Hz, 1H, H-1^(B)),2.50-2.69 (m, 2H, S—CH₂—), 1.37 (d, J_(5,6)=6.2 Hz, 3H, H-6^(B)), 1.33(d, J_(5,6)=6.2 Hz, 3H, H-6^(A)), 1.28 ppm (t, J=7.3 Hz, 3H, S—CH₂—CH₃);¹³C NMR (126 MHz, CDCl₃): δ 165.3, 137.3, 137.1, 133.3, 129.9, 129.7,128.6, 128.5, 128.4 (×2), 128.2, 128.1, 127.9, 99.6, 83.3, 78.1, 76.6,75.3, 72.3, 71.4, 67.7, 67.6, 64.4, 64.1, 25.6, 18.7, 18.5, 14.9 ppm;HRMS (ESI): m/z calcd for C₃₅H₄₀N₆O₇SNa [M+Na]⁺: 711.2571. found:711.2570.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (67)

A mixture of glycosyl donor 53 (0.292 g, 0.540 mmol), glycosyl acceptor65 (Saksena et al. (2008) Carbohydr. Res. 343, 1693-1706) (0.200 g,0.491 mmol) and freshly activated molecular sieves (3 Å, 0.6 g) inCH₂Cl₂ (8 mL) was stirred under argon for 5 h at room temperature.TMSOTf (20 μL, 0.108 mmol) was added and the resulting mixture wasstirred for an additional hour. Then Et₃N (1 mL) was added, the solidwas filtered off and the residue was rinsed with CH₂Cl₂ (3×20 mL). Thecombined filtrate (80 mL) was washed with 20% aq. NaHCO₃ (40 mL), water(30 mL), and brine (20 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.374 g, 97%) as a white foam. Analyticaldata for 67: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D)²′=−41.2 (c=1.6, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.29-8.06 (m, 15H,H—Ar), 5.72 (dd, J_(2,3)=3.3 Hz, 1H, H-2^(B)), 5.66 (dd, J_(3,4)=10.3Hz, 1H, H-3^(B)), 5.29 (d, J_(1,2)=1.8 Hz, 1H, H-1^(B)), 4.82 (d,J_(1,2)=1.8 Hz, 1H, H-1^(A)), 4.73 3.66 (s, 3H, —OCH₃), 1.37 (d,J_(5,6)=6.0 Hz, 3H, H-6^(B)), 1.36 (d, J_(5,6)=6.0 Hz, 3H, H-6^(A)); ¹³CNMR (126 MHz, CDCl₃): δ 174.0, 165.2, 165.1, 137.6, 133.5, 133.3, 129.8,129.4, 129.3, 128.6, 128.5, 128.4, 127.9, 127.6, 99.2, 97.0, 78.6, 72.5,70.7, 70.0, 67.7, 67.6 (×2), 64.7, 63.4, 51.5, 33.9, 29.0, 25.7, 24.6,18.6, 18.5 ppm; HRMS (ESI): m/z calcd for C₄₀H₄₆N₆O₁₁Na [M+Na]⁺:809.3117. found: 809.3107.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (68)

A mixture of glycosyl donor 63 (0.151 g, 0.216 mmol), glycosyl acceptor65 (Saksena et al. (2008) Carbohydr. Res. 343, 1693-1706) (0.080 g,0.196 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) inCH₂Cl₂ (4 mL) was stirred under argon for 5 h at room temperature. MeOTf(133 μL, 1.17 mmol) was added and stirring was continued for additional48 h. Then Et₃N (1 mL) was added, the solid was filtered off and theresidue was rinsed with CH₂Cl₂ (3×20 mL). The combined filtrate (70 mL)was washed with 20% aq. NaHCO₃ (40 mL), water (30 mL), and brine (20mL). The organic phase was separated, dried over MgSO₄, and concentratedin vacuo. The residue was purified by column chromatography on silicagel (ethyl acetate-toluene gradient elution) to afford the titlecompound (0.182 g, 89%) as a white foam. Analytical data for 68: Rf=0.40(ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D) ²¹=−27.7 (c=2.4, CHCl₃);¹H NMR (600 MHz, CDCl₃): δ 7.06-8.01 (m, 20H, H—Ar), 5.71 (dd,J_(2,3)=3.3 Hz, 1H, H-2^(C)), 5.58 (dd, J_(3,4)=10.4 Hz, 1H, H-3^(C)),5.07 (s, 2H, H-1^(B), H-1^(C)), 4.78 (d, J_(1,2)=1.5 Hz, 1H, H-1^(A)),1.46 (d, J_(5,6)=6.2 Hz, 3H, H-6^(C)), 1.34 (d, J_(5,6)=6.5 Hz, 3H,H-6^(B)), 1.32 ppm (d, J_(5,6)=6.2 Hz, 3H, H-6^(A)); ¹³C NMR (126 MHz,CDCl₃): δ 174.0, 165.2, 164.9, 137.6, 137.4, 133.4, 133.2, 129.8, 129.7,129.5, 129.3, 128.5 (×2), 128.4, 128.2, 127.9, 127.8, 127.7, 127.6,101.0, 99.2, 97.0, 78.2, 77.8, 73.4, 72.6, 72.1, 70.9, 69.3, 68.2, 67.7,67.3, 64.9, 63.7, 63.4, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6 (×2), 18.5ppm; HRMS (ESI): m/z calcd for C₅₃H₆₁N₉O₁₄Na [M+Na]⁺: 1070.4230. found:1070.4233.

5′-Methoxycarbonylpentyl4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (69)

A mixture of glycosyl donor 53 (0.168 g, 0.318 mmol), glycosyl acceptor66 Saksena et al. (2005) Tetrahedron: Asymmetry 16, 187-197) (0.118 g,0.289 mmol) and freshly activated molecular sieves (3 Å, 0.350 g) inPhMe (3 mL) was stirred under argon for 2 h at room temperature. Then itwas heated to 95° C. and TMSOTf (12 μL, 0.064 mmol) was added, and themixture stirred for additional 1 h. Then Et₃N (1 mL) was added, thesolid was filtered off and the residue was rinsed with CH₂Cl₂ (3×30 mL).The combined filtrate (100 mL) was washed with 20% aq. NaHCO₃ (50 mL),water (30 mL), and brine (30 mL). The organic phase was separated, driedover MgSO₄, and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (ethyl acetate-toluene gradientelution) to afford the title compound (0.203 g, 91%) as a white foam.Analytical data for 69: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v);[α]_(D) ²¹=−4.4 (c=1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.00-8.06 (m,15H, H—Ar), 5.30 (dd, J_(3,4)=9.9 Hz, 1H, H-3^(B)), 5.07 (d, J_(1,2)=1.7Hz, 1H, H-1^(B)), 4.70 (d, J_(1,2)=1.8 Hz, 1H, H-1^(A)), 3.69 (s, 3H,—OCH₃), 1.39 (d, J=5.9 Hz, 3H, H-6^(A)), 1.34 ppm (d, J=5.7 Hz, 3H,H-6^(B)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 165.4, 137.4, 137.3, 133.2,129.8, 129.5, 128.5, 128.4, 128.2, 128.1, 127.8, 127.6, 99.1, 98.8,78.6, 74.4, 73.3, 72.8, 72.6, 72.5, 67.6, 67.4, 67.3, 64.3, 63.1, 51.5,33.9, 29.1, 25.7, 24.7, 18.5 (×2) ppm; HRMS (ESI): m/z calcd forC₄₀H₄₈N₆O₁₀Na [M+Na]⁺: 795.3324. found: 795.3314.

5′-Methoxycarbonylpentyl4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (70)

Sodium methoxide (˜0.5 mL, 0.5 M solution) was added to a solution of 69(0.910 g, 1.178 mmol) in CH₃OH (20 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.755 g, 96%) as oil. Analytical data for 70:Rf=0.50 (ethyl acetate/toluene, 1/9, v/v); [α]_(D) ²¹=+43.2 (c=1.2,CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.11-7.41 (m, 10H, H—Ar), 5.11 (d,J_(1,2)=1.3 Hz, 1H, H-1^(B)), 4.65 (d, J_(1,2)=2.0 Hz, 1H, H-1^(A)),3.68 (s, 3H, —OCH₃), 1.30 (d, J_(5,6)=6.5 Hz, 3H, H-6^(A)), 1.29 ppm (d,J_(5,6)=6.2 Hz, 3H, H-6^(B)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 137.3,137.1, 128.6, 128.5, 128.2, 128.1, 128.0, 98.8, 97.9, 78.5, 76.5, 72.9,72.6, 72.1, 69.8, 67.5, 67.2, 67.1, 66.4, 64.6, 51.5, 33.9, 29.0, 25.7,24.7, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C₃₃H₄₄N₆O₉Na [M+Na]⁺:691.3062. found: 691.3054.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (71)

A mixture of glycosyl donor 53 (0.124 g, 0.230 mmol), glycosyl acceptor70 (0.140 g, 0.210 mmol) and freshly activated molecular sieves (3 Å,0.5 g) in CH₂Cl₂ (4 mL) was stirred under argon for 5 h at roomtemperature. TMSOTf (8 μL, 0.046 mmol) was added and the resultingmixture was stirred for an additional hour. Then Et₃N (1 mL) was added,the solid was filtered off and the residue was rinsed with CH₂Cl₂ (3×20mL). The combined filtrate (80 mL) was washed with 20% aq. NaHCO₃ (50mL), water (30 mL), and brine (20 mL). The organic phase was separated,dried over MgSO₄, and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (ethyl acetate-toluene gradientelution) to afford the title compound (0.210 g, 96%) as a white foam.Analytical data for 71: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v);[α]_(D) ²¹=−29.0 (c=1.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.22-8.07(m, 20H, H—Ar), 5.70 (dd, J_(2,3)=3.4 Hz, 1H, H-2^(C)), 5.59 (dd,J_(3,4)=9.8 Hz, 1H, H-3^(C)), 5.26 (d, J_(1,2)=1.8 Hz, 1H, H-1^(C)),5.10 (d, J_(1,2)=1.6 Hz, 1H, H-1^(B)), 4.67 (d, J_(1,2)=1.8 Hz, 1H,H-1^(A)), 3.68 (s, 3H, —OCH₃), 1.35 (d, J_(5,6)=6.0 Hz, 3H, H-6^(C)),1.29 (d, J_(5,6)=6.0 Hz, 3H, H-6^(A)), 1.28 ppm (d, J_(5,6)=6.0 Hz, 3H,H-6^(B)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 165.2, 165.0, 137.4, 137.3,133.4, 133.2, 129.8 (×2), 129.4, 129.3, 128.6, 128.5 (×2), 128.3, 128.2,128.1, 127.8, 127.6, 99.1, 98.8, 98.4, 78.4, 77.6, 76.1, 73.4, 72.4,71.8, 70.7, 69.8, 68.2, 67.7, 67.5, 67.1, 64.7, 64.5, 63.3, 51.5, 33.9,29.0, 25.7, 24.7, 18.6 (×2), 18.5 ppm; HRMS (ESI): m/z calcd forC₅₃H₆₁N₉O₁₄Na [M+Na]⁺: 1070.4230. found: 1070.4249.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (72)

A mixture of glycosyl donor 63 (0.208 g, 0.296 mmol), glycosyl acceptor70 (0.180 g, 0.269 mmol) and freshly activated molecular sieves (3 Å,0.5 g) in CH₂Cl₂ (4 mL) was stirred under argon for 4 h at roomtemperature. MeOTf (213 μL, 1.88 mmol) was added and continued stirringfor additional 48 h. Then Et₃N (1 mL) was added, the solid was filteredoff and the residue was rinsed with CH₂Cl₂ (3×20 mL). The combinedfiltrate (70 mL) was washed with 20% aq. NaHCO₃ (40 mL), water (30 mL),and brine (20 mL). The organic phase was separated, dried over MgSO₄,and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.321 g, 91%) as a white foam. Analyticaldata for 72: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D)²¹=−17.9 (c=1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.05-8.00 (m, 25H,H—Ar), 5.71 (dd, J_(2,3)=3.3 Hz, 1H, H-2^(D)), 5.58 (dd, J_(3,4)=10.3Hz, 1H, H-3^(D)), 5.09 (d, J_(1,2)=1.5 Hz, 1H, H-1^(B)), 5.07 (d,J_(1,2)=1.6 Hz, 1H, H-1^(C)), 5.04 (d, J_(1,2)=1.6 Hz, 1H, H-1^(D)),1.31-1.35 (m, 6H, H-6^(A), H-6^(B)), 1.26 ppm (d, J_(5,6)=6.2 Hz, 3H,H-6^(C)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 165.2, 164.8, 137.5, 137.4,137.3, 133.3, 133.2, 129.8, 129.7, 129.5, 129.3, 128.5 (×2), 128.4,128.3, 128.2, 128.0, 127.8 (×2), 127.7, 127.6, 100.8, 99.2, 98.8, 98.3,78.4, 77.9, 77.4, 76.4, 73.7, 73.2, 72.5, 72.1, 71.9, 70.9, 69.3, 68.1,67.9, 67.7, 67.5, 67.2, 64.6, 63.6, 63.4, 51.5, 33.9, 29.1, 25.7, 24.7,18.6 (×2), 18.5 ppm; HRMS (ESI): m/z calcd for C₆₆H₇₆N₁₂O₁₇Na [M+Na]⁺:1331.5344. found: 1331.5341.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (67a)

Sodium methoxide (0.2 mL, 0.5 M solution) was added to a solution of 67(0.350 g, 0.445 mmol) in CH₃OH (5 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.221 g, 86%) as a white foam. Analyticaldata for 67a: Rf=0.50 (CH₃OH/CH₂Cl₂, 0.5/9.5, v/v); [α]_(D) ²¹=+73.4(c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.28-7.41 (m, 5H, H—Ar), 5.07(s, 1H, H-1^(B)), 4.78 (s, 1H, H-1^(A)), 1.32 (d, J_(5,6)=6.2 Hz, 3H,H-6^(A)), 1.27 ppm (d, J_(5,6)=6.2 Hz, 3H, H-6^(B)); ¹³C NMR (126 MHz,CDCl₃): δ 174.1, 137.6, 128.5, 127.9, 127.6, 101.5, 96.9, 78.3, 76.9,72.4, 70.3, 70.2, 67.7, 67.5, 67.3, 65.7, 64.7, 51.5, 33.9, 29.0, 25.7,24.6, 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C₂₆H₃₈N₆O₉Na [M+Na]⁺:601.2592. found: 601.2581.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (44)

To a stirred solution of 67a (0.150 g, 0.259 mmol), in a pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., and thenstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 67b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₂₆H₄₃N₂O₉ [M+H]⁺: 527.2963. found: 527.2964.This crude material was directly used for formylation.

Compound 67b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydride²² (5 mL, ethereal solution) and stirred for 3 h, thenslowly allowed to warm to room temperature. Then solvents wereevaporated and the residue was passed through column chromatography onsilica gel (methanol-dichloromethane gradient elution) to afforddisaccharide 67c. HRMS (ESI): m/z calcd for C₂₈H₄₂N₂NaO₁₁ [M+Na]⁺:605.2681. found: 605.2675.

Compound 67c was dissolved in CH₃OH/H₂O (2:1, 6 mL), Pd(OH)₂ on carbon(20%, 0.050 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.075 g, 59%, over 3 steps) as a white foam. Analytical data for 44:Rf=0.20 (CH₃OH/CH₂Cl₂, 1.5/8.5, v/v); ¹H NMR (700 MHz, D₂O): δ 8.21 ((d,J=15.4 Hz) and 8.03 (d, J=13.3 Hz), 2H, NCHO), 4.81-4.95 (m, 2H, 2×H-1),3.70 (s, 3H, —OCH₃), 1.20-1.30 ppm (m, 6H, 2×H-6); ¹³C NMR (126 MHz,D₂O): δ 178.5, 168.9, 168.8, 165.8, 165.7, 103.3, 103.2, 100.6, 100.5,77.8 (×2), 70.2, 70.0, 69.9 (×2), 69.0, 68.9 (×2), 68.8 (×2), 68.6,68.4, 68.2, 67.8, 57.6, 56.5, 53.1, 52.7, 52.6, 51.6, 34.6, 29.1, 25.9,25.0, 17.8, 17.7, 17.6 ppm; HRMS (ESI): m/z calcd for C₂H₃₆N₂O₁₁Na[M+Na]⁺: 515.2211. found: 515.2210.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranoside (81)

A solution of 44 (0.009 g, 0.018 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on C18 column in gradient water-acetonitrile and lyophilized, togive the title compound (0.0075 g, 79%) as a white foam. Analytical datafor 81: Rf=0.15 (CH₃OH/CH₂Cl₂, 1/1, v/v); ¹H NMR (700 MHz, D₂O): δ8.19-8.22 (Z) and 8.01-8.02 (F) (m, 2H, NCHO), 4.80-4.95 (m, 2H, 2×H-1),1.19-1.30 ppm (m, 6H, 2×H-6); ¹³C NMR (126 MHz, D₂O): δ 178.4, 168.9,168.8, 165.8, 165.7, 103.6, 103.3 (×2), 100.6, 100.5, 77.8, 77.4, 71.1,70.4, 70.2, 70.0, 69.8, 69.0, 68.9, 68.8 (×3), 68.6, 68.4, 68.2, 67.8,57.6, 56.5, 54.5, 52.6, 51.8, 51.6, 41.7, 41.4, 40.9, 40.7, 36.7, 29.2,26.0, 25.9, 17.9, 17.8, 17.7 (×2) ppm; HRMS (ESI): m/z calcd forC₂₂H₄₀N₄O₁₀Na [M+Na]⁺: 543.2637. found: 543.2642.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(87)

To a stirred solution of 81 (0.0075 g, 0.014 mmol) in water (0.5 mL) andEtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20%in ethanol, 70 μL) was added and the pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.0089g, 92%) as a white foam. Analytical data for 87: Rf=0.20 (CH₃OH/CH₂Cl₂,1.5/8.5, v/v); ¹H NMR (500 MHz, D₂O): δ 8.26-8.30 (Z) and 8.09-8.12 (E)(m, 2H, NCHO), 4.83-5.03 (m, 2H, 2×H-1), 1.26-1.37 (m, 6H, 2×H-6),0.99-1.05 ppm (m, 3H, —CH₃₁); ¹³C NMR (126 MHz, D₂O): δ 189.8, 189.6,184.3 (×2), 178.5, 178.1, 178.0, 177.9, 174.8, 174.7, 168.9, 168.8,165.8, 165.7, 103.3, 103.2, 100.5 (×2), 77.8, 75.4, 75.3, 70.2, 70.0,69.9, 69.0, 68.9 (×2), 68.8, 68.7, 68.6, 68.4, 68.2, 67.7, 57.6, 56.5,52.7, 52.6, 51.7, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 31.2, 29.3 (×2),26.2, 26.1, 25.9 (×2), 19.1, 19.0, 17.8 (×2), 17.7, 13.9 ppm; HRMS(ESI): m/z calcd for C₃₀H₄₈N₄O₁₃Na [M+Na]⁺: 695.3110. found: 695.3113.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (68a)

Sodium methoxide (0.2 mL, 0.5 M solution) was added to a solution of 68(0.352 g, 0.336 mmol) in CH₃OH (5 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.256 g, 91%) as an off-white foam.Analytical data for 68a: Rf=0.30 (ethyl acetate/toluene, 1/4, v/v);[α]_(D) ²¹=+72.4 (c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.27-7.39(m, 10H, H—Ar), 5.02 (s, 1H, H-1^(B)), 4.93 (s, 1H, H-1^(C)), 4.77 (s,1H, H-1^(A)), 3.67 (s, 3H, —OCH₃), 1.24 ppm (d, J_(5,6)=6.2 Hz, 3H,H-6^(C)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 137.6, 137.4, 128.6, 128.5,128.1, 127.9, 127.5, 101.0 (×2), 97.0, 78.3, 77.8, 73.1, 72.5, 72.1,70.2, 69.9, 67.9, 67.6, 67.4, 67.3, 65.8, 64.8, 64.0, 51.5, 33.9, 29.0,25.6, 24.6, 18.6, 18.5, 18.3 ppm; HRMS (ESI): m/z calcd forC₃₉H₅₃N₉O₁₂Na [M+Na]⁺: 862.3706. found: 862.3691.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranoside (44)

To a stirred solution of 68a (0.114 g, 0.136 mmol), in pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., and thenstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 68b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₃₉H₆₀N₃O₁₂ [M+H]+: 762.4172. found: 762.4171.This crude material was directly used for formylation.

Compound 68b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydrid²² (5 mL, ethereal solution) and stirred for 3 h, thenslowly allowed to warm to room temperature. Then solvents wereevaporated and the residue was passed through column chromatography onsilica gel (methanol-dichloromethane gradient elution) to afforddisaccharide 68c. HRMS (ESI): m/z calcd for C₄₂H₅₉N₃O₁₅Na [M+Na]⁺:868.3838. found: 868.3827.

Compound 68c was dissolved in CH₃OH/H₂O (2:1, 5 mL), Pd(OH)₂ on carbon(20%, 0.040 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.046 g, 51%, over 3 steps) as a white foam. Analytical data for 45:Rf=0.40 (CH₃OH/CH₂Cl₂, 3/7, v/v); ¹H NMR (700 MHz, D₂O): δ 8.20-8.24 (Z)and 8.02-8.06 (E) (m, 3H, NCHO), 4.82-5.08 (m, 3H, 3×H-1), 1.21-1.32 ppm(m, 9H, 3×H-6); ¹³C NMR (126 MHz, D₂O): δ 178.5, 168.8, 168.7, 165.8(×2), 165.6 (×2), 103.4, 103.3, 102.9, 101.8, 101.7, 100.6, 100.5, 79.0(×2), 78.9, 78.2, 78.0, 77.7 (×3), 70.0, 69.9, 69.8, 69.4, 69.2, 69.0,68.9 (×2), 68.8 (×2), 68.7, 68.6 (×2), 68.5 (×2), 68.3 (×2), 67.9, 67.7,57.8, 57.7, 56.4, 53.1, 52.8, 52.7, 51.9, 34.6, 29.1, 25.9, 25.0, 18.1,17.9 (×3), 17.8, 17.7 ppm; HRMS (ESI): m/z calcd for C₂₈H₄₇N₃O₁₅Na[M+Na]⁺: 688.2899. found: 688.2895.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranoside (82)

A solution of 45 (0.012 g, 0.018 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on a C18 column with a gradient of water-acetonitrile andlyophilized, to give the title compound (0.0112 g, 90%) as a white foam.Analytical data for 82: Rf=0.10 (CH₃OH/CH₂Cl₂, 1/1, v/v); ¹H NMR (500MHz, D₂O): δ 8.26-8.31 (Z) and 8.08-8.13 (E) (m, 3H, NCHO), 4.88-5.16(m, 3H, 3×H-1), 1.20-1.37 (m, 9H, 3×H-6); ¹³C NMR (126 MHz, D₂O): δ178.4, 178.2, 168.8 (×2), 165.9, 165.8, 165.6 (×3), 103.3 (×2), 101.7(×2), 100.5 (×2), 79.0, 77.7, 70.0, 69.9, 69.2, 69.0, 68.9, 68.8, 68.6,68.4, 68.0, 67.7, 57.8, 57.7, 52.8, 52.7, 51.9, 41.5 (×2), 41.5, 41.4,40.9, 40.6 (×2), 36.6, 29.2, 26.0 (×2), 25.9, 25.8, 18.1, 18.0, 17.9(×3), 17.7 ppm; HRMS (ESI): m/z calcd for C₂₉H₅₁N₅O₁₄Na [M+Na]⁺:716.3325, found: 716.3311.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(88)

To a stirred solution of 82 (0.0075 g, 0.011 mmol) in water (0.5 mL) andEtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20%in ethanol, 50 μL) was added and the pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.0065g, 71%) as a white foam. Analytical data for 88: Rf=0.20 (CH₃OH/CH₂Cl₂,1/4, v/v); ¹H NMR (500 MHz, D₂O): δ 8.27-8.31 (Z) and 8.08-8.13 (E) (m,3H, NCHO), 4.86-5.15 (m, 3H, 3×H-1), 1.26-1.36 (m, 9H, 3×H-6), 0.98-1.05ppm (m, 3H, —CH₃₁); ¹³C NMR (126 MHz, D₂O): δ 189.8, 189.6, 184.3 (×2),178.5, 178.1, 178.0, 177.9, 174.8, 174.7, 168.8, 168.7, 165.8 (×2),165.6, 103.4, 103.3, 102.9, 101.8, 101.7, 100.6, 100.5, 79.0, 78.9,78.8, 78.2, 78.0, 77.7 (×2), 75.4, 75.3, 70.0, 69.9, 69.3, 69.2, 69.0,68.9, 68.9, 68.8, 68.6, 68.5 (×2), 68.4, 68.3, 67.9, 67.7, 57.8, 57.7,56.5, 52.8, 52.8, 52.7, 51.9, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 29.3(×2), 26.2, 26.1, 25.9 (×2), 19.1, 19.0, 18.1, 18.0, 17.9 (×3), 17.7,13.9 ppm; HRMS (ESI): m/z calcd for C₃₇H₅₉N₅O₁₇Na [M+Na]⁺: 868.3798.found: 868.3800.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (71a)

Sodium methoxide (0.5 mL, 0.5 M solution) was added to a solution of 71(0.458 g, 0.437 mmol) in CH₃OH (10 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.341 g, 93%) as oil. Analytical data for71a: Rf=0.30 (ethyl acetate/toluene, 1/4, v/v); [α]_(D) ²¹=+58.9 (c=1.3,CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.12-7.39 (m, 10H, H—Ar), 5.08 (d,J_(1,2)=1.8 Hz, 1H, H-1^(B)), 5.05 (d, J_(1,2)=1.6 Hz, 1H, H-1^(C)),4.66 (d, J_(1,2)=1.6 Hz, 1H, H-1^(A)), 3.68 (s, 3H, —OCH₃), 1.28-1.32(m, 6H, H-6^(A), H-6^(B)), 1.19 ppm (d, J_(5,6)=6.2 Hz, 3H, H-6^(C));¹³C NMR (126 MHz, CDCl₃): δ 174.1, 137.3 (×2), 128.5, 128.4, 128.2,128.1, 127.8, 127.6, 101.5, 98.8, 98.1, 78.4, 77.5, 76.4, 73.2, 72.6,71.7, 70.1, 70.0, 67.9, 67.5, 67.4, 67.2, 65.5, 64.7, 64.4, 51.5, 33.9,29.0, 25.7, 24.6, 18.6, 18.5, 18.2 ppm; HRMS (ESI): m/z calcd forC₃₉H₅₃N₉O₁₂Na [M+Na]⁺: 862.3706. found: 862.3700.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (46)

To a stirred solution of 71a (0.157 g, 0.187 mmol), in pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., and thenstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 71b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₃₉H₆₀N₃O₁₂ [M+H]+: 762.4172. found: 762.4182.This crude material was directly used for formylation.

Compound 71b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydride²² (5 mL, ethereal solution) and stirred for 3 h, thenslowly allowed to warm to room temperature. Then solvents wereevaporated and the residue was passed through column chromatography onsilica gel (methanol-dichloromethane gradient elution) to afforddisaccharide 71c. HRMS (ESI): m/z calcd for C₄₂H₅₉N₃O₁₅Na [M+Na]⁺:868.3838. found: 868.3834.

Compound 71c was dissolved in CH₃OH/H₂O (2:1, 5 mL), Pd(OH)₂ on carbon(20%, 0.050 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.053 g, 43%, over 3 steps) as a white foam. Analytical data for 46:Rf=0.50 (CH₃OH/CH₂Cl₂, 3/7, v/v); ¹H NMR (700 MHz, D₂O): δ 8.20-8.24 (Z)and 8.02-8.06 (F) (m, 3H, NCHO), 4.92-5.04 (m, 3H, 3×H-1), 3.70 (s, 3H,—OCH₃), 1.22-1.31 ppm (m, 9H, 3×H-6); ¹³C NMR (176 MHz, D₂O): δ 178.3,168.7, 168.6, 168.5, 165.6 (×2), 165.5, 103.1, 102.9 (×2), 102.8, 102.7(×2), 99.0 (×3), 78.6 (×2), 78.5, 77.3, 77.2, 77.1, 70.1, 70.0, 69.8,69.5, 69.4, 68.9, 68.8 (×2), 68.7 (×2), 68.6 (×2), 68.5 (×2), 68.4 (×2),68.3, 68.2, 67.8, 57.7, 57.4, 56.3, 52.9, 52.8, 52.4, 51.3, 34.4, 28.9,25.7, 24.8, 17.6 (×2), 17.5, 17.4 ppm; HRMS (ESI): m/z calcd forC₂₈H₄₇N₃O₁₅Na [M+Na]⁺: 688.2899. found: 688.2893.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (83)

A solution of 46 (0.010 g, 0.015 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on a C18 column with a gradient of water-acetonitrile andlyophilized, to give the title compound (0.0084 g, 81%) as a white foam.Analytical data for 83: Rf=0.10 (CH₃OH/CH₂Cl₂, 1/1, v/v); ¹H NMR (500MHz, D₂O): δ 8.26-8.31 (Z) and 8.09-8.13 (E) (m, 3H, NCHO), 4.98-5.11(m, 3H, 3×H-1), 1.26-1.38 ppm (m, 9H, 3×H-6); ¹³C NMR (126 MHz, D₂O): δ177.7, 177.2, 168.0, 167.9, 167.8, 164.9, 164.8, 164.6, 102.4, 102.2(×2), 102.1, 101.9 (×2), 98.3, 98.2, 77.9, 77.8 (×3), 76.8, 76.5 (×2),76.4, 76.2, 70.0, 69.9, 69.5, 69.3, 69.0, 68.8 (×2), 68.7, 68.2, 68.1,68.0 (×2), 67.9, 67.8 (×2), 67.7, 67.6 (×2), 67.5, 67.1, 57.0, 56.7,55.5, 53.5, 52.0, 51.7 (×2), 51.6, 50.7, 50.6 (×2), 40.4, 39.9, 39.5,39.0, 35.7, 35.6, 28.2, 25.1, 24.9 (×2), 17.0, 16.9, 16.7 (×2) ppm; HRMS(ESI): m/z calcd for C₂₉H₅₁N₅O₁₄Na [M+Na]⁺: 716.3325. found: 716.3322.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(89)

To a stirred solution of 83 (0.0074 g, 0.0106 mmol) in water (0.5 mL)and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione(20% in ethanol, 50 μL) was added and pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.0072g, 80%) as a white foam. Analytical data for 89: Rf=0.20 (CH₃OH/CH₂Cl₂,1/4, v/v); ¹H NMR (500 MHz, D₂O): δ 8.16-8.21 (Z) and 7.99-8.03 (E) (m,3H, NCHO), 4.87-5.01 (m, 3H, 3×H-1), 1.18-1.27 (m, 9H, 3×H-6); ¹³C NMR(126 MHz, D₂O): δ 189.8, 189.6, 184.2, 178.5, 178.0, 177.9 (×2), 174.8,174.7, 168.8, 168.7, 166.6, 165.8, 165.7, 103.1, 102.8, 99.1 (×2), 78.7,77.4 (×2), 75.4, 75.3, 70.2, 69.7, 69.6, 69.1, 69.0, 68.9, 68.7 (×3),68.5, 68.4, 53.0, 52.6, 51.5, 45.1, 44.9, 40.3, 40.1, 36.7, 32.4, 29.2,26.1, 26.0, 25.9 (×2), 19.0 (×2), 17.8 (×3), 17.6, 13.9 ppm; HRMS (ESI):m/z calcd for C₃₇H₅₉N₅O₁₇Na [M+Na]⁺: 868.3798. found: 868.3791.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (72a)

Sodium methoxide (0.5 mL, 0.5 M solution) was added to a solution of 72(0.391 g, 0.299 mmol) in CH₃OH/THF mixture (4:1, 15 mL) until pH˜9 andthe resulting mixture was stirred under argon for 5 h at roomtemperature. Then the reaction mixture was neutralized with Amberlite IR120 (H⁺) ion exchange resin, the resin was filtered off and rinsedsuccessively with CH₃OH. The combined filtrate was concentrated in vacuoand purified by column chromatography on silica gel (ethylacetate-toluene gradient elution) to afford the title compound (0.311 g,95%) as oil. Analytical data for 72a: Rf=0.40 (ethyl acetate/toluene,1/4, v/v); [α]_(D) ²¹=+51.2 (c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ7.10-7.39 (m, 15H, H—Ar), 5.07 (d, J_(1,2)=1.6 Hz, 1H, H-1^(B)), 5.02(d, J_(1,2)=1.6 Hz, 1H, H-1^(C)), 4.91 (d, J_(1,2)=1.5 Hz, 1H, H-1^(D)),4.66 (d, J_(1,2)=2.0 Hz, 1H, H-1^(A)), 3.68 (s, 3H, —OCH₃), 1.26-1.35(m, 9H, H-6^(A), H-6^(B) H-6^(C)), 1.17 ppm (d, J_(5,6)=5.9 Hz, 3H,H-6^(D)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 137.5, 137.4, 137.3, 128.5,128.4, 128.2, 128.0 (×2), 127.8, 127.5, 101.0, 100.8, 98.8, 98.2, 78.4,77.9, 77.5, 76.4, 73.2, 72.5, 72.1, 71.8, 70.0, 69.7, 67.9 (×2), 67.5(×2), 67.1, 65.8 (×2), 64.7, 64.6, 63.9, 51.5, 33.9, 29.0, 25.7, 24.7,18.6 (×2), 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C₅₂H₆₈N₁₂O₁₅Na[M+Na]⁺: 1123.4806. found: 1123.4812.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (47)

To a stirred solution of 72a (0.146 g, 0.132 mmol), in pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., and thenstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 72b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₅₂H₇₇N₄O₁₅ [M+H]+: 997.5380. found: 997.5366.This crude material was directly used for formylation.

Compound 72b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydride Olah et al. (Angew (1979) Chem. Int. Ed. 18, 614) (5mL, ethereal solution) and stirred for 3 h, then slowly allowed to warmto room temperature. Then solvents were evaporated and the residue waspassed through column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford disaccharide 72c.HRMS (ESI): m/z calcd for C₅₆H₇₆N₄O₁₉Na [M+Na]⁺: 1131.4996. found:1131.4992.

Compound 72c was dissolved in CH₃OH/H₂O (2:1, 5 mL), Pd(OH)₂ on carbon(20%, 0.050 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.068 g, 61%, over 3 steps) as a white foam. Analytical data for 47:Rf=0.30 (CH₃OH/CH₂Cl₂, 3/7, v/v); ¹H NMR (500 MHz, D₂O): δ 8.26-8.33 (Z)and 8.06-8.14 (E) (m, 4H, NCHO), 4.98-5.20 (m, 4H, 4×H-1), 3.85-4.28 (m,16H, 4×H-2, 4×H-3, 4×H-4, 4×H-5), 1.28-1.39 ppm (m, 12H, 4×H-6); ¹³C NMR(126 MHz, D₂O): δ 178.5, 168.8, 168.7, 168.6, 165.8, 165.6, 103.3,103.2, 102.9, 102.8, 102.7, 101.9, 101.6, 99.3, 99.2, 79.3, 78.9, 78.7,78.5, 78.4, 78.1, 77.3, 69.9 (×2), 69.8, 69.7, 69.4, 69.3, 69.0, 68.9,68.8 (×2), 68.7, 68.6, 68.5, 68.4, 68.3, 68.0, 58.0, 57.7, 56.5, 53.1,52.9, 52.8, 52.7, 51.8, 34.6, 29.1, 25.9, 25.0, 18.1 (×2), 18.0 (×2),17.9 (×2), 17.8 (×2), 17.7 (×2) ppm; HRMS (ESI): m/z calcd forC₃₅H₅₈N₄O₁₉Na [M+Na]⁺: 861.3587. found: 861.3580.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (84)

A solution of 47 (0.0134 g, 0.016 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on a C18 column with a gradient of water-acetonitrile andlyophilized, to give the title compound (0.0113 g, 82%) as a white foam.Analytical data for 84: Rf=0.10 (CH₃OH/CH₂Cl₂, 1/1, v/v); ¹H NMR (500MHz, D₂O): δ 8.16-8.23 (Z) and 7.98-8.05 (E) (m, 4H, NCHO), 4.84-5.10(m, 4H, 4×H-1), 2.22-2.28 (m, 2H, —CH_(2f)—), 1.54-1.64 (m, 4H,—CH_(2e)—, —CH_(2c)), 1.30-1.41 (m, 2H, —CH_(2d)—), 1.18-1.30 ppm (m,12H, 4×H-6); ¹³C NMR (125 MHz, D₂O): δ 177.4, 167.9 (×2), 164.9, 164.6,102.6, 102.3 (×3), 101.9 (×2), 101.0 (×2), 100.8, 98.6, 98.3, 78.1,77.6, 77.5 (×2), 76.8, 76.4, 76.0, 70.3, 70.2, 70.1, 70.0 (×2), 69.5,69.2, 68.9 (×3), 68.3, 68.0, 67.8, 67.6 (×2), 53.9, 53.8, 53.6, 52.0,51.8, 50.8 (×2), 40.6 (×3), 39.7, 35.7, 28.2, 25.0, 24.9 (×2), 17.3,17.0, 16.8 (×3) ppm; HRMS (ESI): m/z calcd for C₃₆H₆₂N₆O₁₈Na [M+Na]⁺:889.4013. found: 889.4020.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(90)

To a stirred solution of 84 (0.013 g, 0.015 mmol) in water (0.5 mL) andEtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20%in ethanol, 70 μL) was added and pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.0109g, 72%) as a white foam. Analytical data for 90: Rf=0.20 (CH₃OH/CH₂Cl₂,1/4, v/v); ¹H NMR (600 MHz, D₂O): δ 8.18-8.24 (Z) and 7.98-8.06 (E) (m,4H, NCHO), 4.90-5.10 (m, 4H, 4×H-1), 1.20-1.35 (m, 14H, —CH_(2d)—,4×H-6) ppm; ¹³C NMR (126 MHz, D₂O): δ 189.8, 189.6, 184.3 (×2), 178.5,178.2, 178.1, 178.0, 177.9, 174.8, 174.7, 168.8, 168.7, 168.6, 165.8,165.6, 103.3 (×2), 103.2, 102.8 (×2), 102.7, 101.7 (×2), 101.6 (×2),99.2, 78.9 (×2), 78.4, 78.1, 77.3, 75.4, 75.3, 69.9 (×2), 69.8 (×2),69.7, 69.3, 69.0 (×2), 68.9, 68.8 (×4), 68.6 (×2), 68.5, 68.4 (×2),68.0, 62.5, 52.9, 52.8 (×2), 52.7, 51.8, 45.2, 45.0, 44.2, 40.6, 40.3,40.2, 36.7, 34.4, 32.4, 29.2, 26.2, 26.1, 26.0 (×2), 25.9 (×2), 25.8,19.3, 19.1, 19.0, 18.1, 18.0 (×2), 17.9, 17.8 (×2), 17.7 (×2), 14.0,13.9 ppm; HRMS (ESI): m/z calcd for C₄₄H₇₀N₆O₂₁Na [M+Na]⁺: 1041.4486.found: 1041.4484.

5′-Methoxycarbonylpentyl4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (73)

A mixture of glycosyl donor 64 (0.743 g, 1.08 mmol), glycosyl acceptor66 Cheng. et al. (2010) Angew. Chem. Int. Ed. 49, 4771-4774) (0.400 g,0.982 mmol) and freshly activated molecular sieves (3 Å, 1.5 g) inCH₂Cl₂ (10 mL) was stirred under argon for 4 h at room temperature.MeOTf (0.890 mL, 7.86 mmol) was added and stirring was continued for anadditional 48 h. Then Et₃N (1 mL) was added, the solid was filtered offand the residue was rinsed with CH₂Cl₂ (3×30 mL). The combined filtrate(100 mL) was washed with 20% aq. NaHCO₃ (40 mL), water (30 mL), andbrine (20 mL). The organic phase was separated, dried over MgSO₄, andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (ethyl acetate-toluene gradient elution) to afford thetitle compound (0.865 g, 85%) as oil. Analytical data for 73: Rf=0.50(ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D) ²¹=+36.7 (c=1.3, CHCl₃);¹H NMR (500 MHz, CDCl₃): δ 7.15-8.11 (m, 20H, H—Ar), 5.61 (dd,J_(2,3)=3.0 Hz, 1H, H-2^(C)), 4.99 (d, J_(1,2)=1.8 Hz, 1H, H-1^(B)),4.90 (d, J_(1,2)=2.0 Hz, 1H, H-1^(C)), 4.64 (s, 1H, H-1^(A)), 3.70 (s,3H, —OCH₃), 1.30 (d, J_(5,6)=6.0 Hz, 6H, H-6^(B), H-6^(C)), 1.25 ppm (d,J_(5,6)=6.0 Hz, 3H, H-6^(A)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 165.3,137.4, 137.3, 137.2, 133.3, 129.9, 129.8, 128.5 (×3), 128.4, 128.3,128.1, 128.0 (×2), 127.9, 100.4, 99.2, 98.7, 77.6, 76.7, 75.4, 74.2,74.1, 72.2, 72.1, 71.4, 67.7 (×2), 67.5, 67.1, 64.4, 64.1 (×2), 51.5,33.9, 29.1, 25.7, 24.7, 18.7, 18.6 ppm; HRMS (ESI): m/z calcd forC₅₃H₆₃N₉O₁₃Na [M+Na]⁺: 1056.4438. found: 1056.4436.

5′-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (74)

Sodium methoxide (0.7 mL, 0.5 M solution) was added to a solution of 73(0.855 g, 0.827 mmol) in CH₃OH (10 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H^(f)) ionexchange resin, the resin was filtered off and rinsed successively withCH₃OH. The combined filtrate was concentrated in vacuo and purified bycolumn chromatography on silica gel (ethyl acetate-toluene gradientelution) to afford the title compound (0.720 g, 94%) as oil. Analyticaldata for 74: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D)²¹+94.4 (c=1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.29-7.45 (m, 15H,H—Ar), 4.95 (br. s., 2H, H-1^(B), H-1^(C)), 4.63 (d, J_(1,2)=2.0 Hz, 1H,H-1^(A)), 3.70 (s, 3H, —OCH₃), 1.28-1.31 (m, 6H, H-6^(B), H-6^(C)), 1.21ppm (d, J_(5,6)=6.2 Hz, 3H, H-6^(A)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0,137.4, 137.3, 137.1, 128.6 (×2), 128.3 (×2), 128.2 (×3), 128.1, 100.5(×2), 98.7, 77.6, 77.5, 76.9, 74.0, 73.3, 72.2, 72.1 (×2), 67.7, 67.5,67.3, 67.2, 67.1, 64.4, 64.2, 63.8, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6(×2), 18.3 ppm; HRMS (ESI): m/z calcd for C₄₆H₅₉N₉O₁₂Na [M+Na]⁺:952.4175. found: 952.4176.

5′-Methoxycarbonylpentyl4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (75)

A mixture of glycosyl donor 64 (0.262 g, 0.381 mmol), glycosyl acceptor74 (0.322 g, 0.346 mmol) and freshly activated molecular sieves (3 Å,0.5 g) in CH₂Cl₂ (8 mL) was stirred under argon for 4 h at roomtemperature. MeOTf (320 μL, 2.77 mmol) was added and stirring wascontinued for an additional 48 h. Then Et₃N (1 mL) was added, the solidwas filtered off and the residue was rinsed with CH₂Cl₂ (3×20 mL). Thecombined filtrate (70 mL) was washed with 20% aq. NaHCO₃ (40 mL), water(30 mL), and brine (20 mL). The organic phase was separated, dried overMgSO₄, and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.461 g, 86%) as a white foam. Analyticaldata for 75: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D)²¹=+52.7 (c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.16-8.09 (m, 30H,H—Ar), 5.60 (dd, J_(2,3)=3.1 Hz, 1H, H-2^(E)), 4.98 (d, J_(1,2)=1.8 Hz,1H, H-1^(D)), 4.92 (d, =1.8 Hz, 1H, H-1^(E)), 4.88 (d, =2.0 Hz, 1H,H-1^(C)), 4.86 (d, J_(1,2)=2.0 Hz, 1H, H-1^(B)), 1.23-1.29 (m, 9H,H-6^(B), H-6^(C) H-6^(D)), 1.19 (d, J_(5,6)=6.2 Hz, 3H, H-6^(E)), 1.15ppm (d, J_(5,6)=6.2 Hz, 3H, H-6^(A)); ¹³C NMR (126 MHz, CDCl₃): δ 173.9,165.3, 137.4, 137.3, 137.1 (×2), 133.3, 129.9, 129.8, 128.7, 128.6 (×3),128.5 (×2), 128.4 (×2), 128.3 (×3), 128.2 (×2), 128.1 (×3), 127.9 (×2),100.4, 100.2, 100.1, 99.2, 98.6, 77.4, 76.6, 75.3, 74.1, 74.0, 73.6,72.2 (×2), 72.1 (×2), 71.3, 67.8 (×2), 67.7 (×2), 67.5, 67.1, 64.4,64.3, 64.2, 64.1 (×2), 51.5, 33.9, 29.0, 25.7, 24.7, 18.6, 18.5 (×2)ppm; HRMS (ESI): m/z calcd for C₇₉H₉₃N₁₅O₁₉Na [M+Na]⁺: 1578.6664. found:1578.6667.

5′-Methoxycarbonylpentyl4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (76)

Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 75(0.450 g, 0.289 mmol) in CH₃OH (10 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.395 g, 94%) as oil. Analytical data for 76:Rf=0.40 (ethyl acetate/toluene, 1/9, v/v); [α]_(D) ²¹=+81.2 (c=1.0,CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.28-7.41 (m, 25H, H—Ar), 4.97 (d,J_(1,2)=1.1 Hz, 1H, H-1^(E)), 4.96 (d, J_(1,2)=1.5 Hz, 1H, H-1^(D)),4.87 (d, J_(1,2)=1.3 Hz, 1H, H-1^(C)), 4.85 (d, J_(1,2)=1.3 Hz, 1H,H-1^(B)), 1.14-1.26 ppm (m, 15H, H-6^(A), H-6^(B), H-6^(C), H-6^(D),H-6^(E)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 137.3 (×2), 137.2, 137.1(×2), 128.6 (×4), 128.4 (×2), 128.3 (×3), 128.2 (×2), 128.1 (×2), 100.5,100.4, 100.2 (×2), 98.6, 77.7, 77.4, 76.6, 76.5, 74.0, 73.6, 73.5, 73.3,72.2 (×2), 72.1 (×2), 67.8, 67.7, 67.5, 67.3, 67.1 (×2), 64.4, 64.2,63.8, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.5 (×2), 18.3 ppm; HRMS(ESI): m/z calcd for C₇₂H₈₉N₁₅O₁₈Na [M+Na]⁺: 1474.6402. found:1474.6406.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (77)

A mixture of glycosyl donor 53 (0.158 g, 0.292 mmol), glycosyl acceptor76 (0.386 g, 0.266 mmol) and freshly activated molecular sieves (3 Å,0.5 g) in CH₂Cl₂ (5 mL) was stirred under argon for 5 h at roomtemperature. TMSOTf (11 μL, 0.058 mmol) was added and the resultingmixture was stirred for an additional hour. Then Et₃N (1 mL) was added,the solid was filtered off and the residue was rinsed with CH₂Cl₂ (3×20mL). The combined filtrate (80 mL) was washed with 20% aq. NaHCO₃ (50mL), water (30 mL), and brine (20 mL). The organic phase was separated,dried over MgSO₄, and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (ethyl acetate-toluene gradientelution) to afford the title compound (0.487 g, 90%) as a white foam.Analytical data for 77: Rf=0.70 (ethyl acetate/toluene, 1/9, v/v);[α]_(D) ²¹=+31.7 (c=1.4, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.13-8.03(m, 35H, H—Ar), 5.70 (dd, J_(2,3)=3.3 Hz, 1H, H-2^(F)), 5.59 (dd,J_(3,4)=10.2 Hz, 1H, H-3^(F)), 5.02-5.03 (m, 2H, H-1^(E), H-1^(F)), 4.90(dd, J_(1,2)=1.8 Hz, 1H, H-1^(D)), 4.88 (dd, J_(1,2)=1.8 Hz, 1H,H-1^(C)), 4.86 (dd, J_(1,2)=1.8 Hz, 1H, H-1^(B)), 1.14-1.31 ppm (m, 18H,H-6^(A), H-6^(B), H-6^(C), H-6^(D), H-6^(E), H-6^(F)); ¹³C NMR (126 MHz,CDCl₃): δ 174.0, 165.2, 164.9, 137.4, 137.3, 137.1 (×3), 133.4, 133.2,129.8, 129.7, 129.5, 129.3, 129.0, 128.7, 128.6 (×3), 128.5 (×2), 128.4(×3), 128.3 (×2), 128.2, 128.1 (×2), 127.9 (×3), 100.4, 100.3, 100.1,100.0, 98.9, 98.6, 77.4, 77.1, 76.6, 76.5, 74.0, 73.8, 73.6, 73.5, 73.0,72.3 (×2), 72.2 (×2), 72.1, 70.9, 69.4, 68.1, 67.9, 67.8 (×2), 67.6,67.5, 67.1, 64.4, 64.3, 64.2 (×2), 63.9, 63.4, 51.5, 33.9, 29.0, 25.7,24.7, 18.6 (×2), 18.5 (×2), 18.4 ppm; HRMS (ESI): m/z calcd forC₉₂H₁₀₆N₁₈O₂₃Na [M+Na]⁺: 1853.7570. found: 1853.7550.

5′-Methoxycarbonylpentyl4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (78)

A mixture of glycosyl donor 58 (0.228 g, 0.433 mmol), glycosyl acceptor74 (0.366 g, 0.394 mmol) and freshly activated molecular sieves (3 Å,0.500 g) in PhMe (10 mL) was stirred under argon for 2 h at roomtemperature. Then it was heated to 95° C. and TMSOTf (16 μL, 0.087 mmol)was added, and the mixture was stirred for an additional 60 min. ThenEt₃N (1 mL) was added, the solid was filtered off and the residue wasrinsed with CH₂Cl₂ (3×30 mL). The combined filtrate (100 mL) was washedwith 20% aq. NaHCO₃ (50 mL), water (30 mL), and brine (30 mL). Theorganic phase was separated, dried over MgSO₄, and concentrated invacuo. The residue was purified by column chromatography on silica gel(ethyl acetate-toluene gradient elution) to afford the title compound(0.444 g, 87%) as a white foam. Analytical data for 78: Rf=0.50 (ethylacetate/toluene, 0.5/9.5, v/v); [α]_(D) ²¹=+42.1 (c=1.0, CHCl₃); ¹H NMR(600 MHz, CDCl₃): δ 7.03-8.05 (m, 25H, H—Ar), 5.29 (dd, J_(3,4)=10.3 Hz,1H, H-3^(D)), 5.07 (d, J_(1,2)=1.6 Hz, 1H, H-1^(D)), 4.96 (d,J_(1,2)=1.8 Hz, 1H, H-1^(C)), 4.91 (d, J_(1,2)=1.8 Hz, 1H, H-1^(B)),1.22-1.29 ppm (m, 12H, H-6^(A), H-6^(B), H-6^(C), H-6^(D)); ¹³C NMR (126MHz, CDCl₃): δ 174.0, 165.4, 137.4, 137.3 (×2), 137.1, 133.3, 129.8,129.5, 128.7, 128.6 (×2), 128.4, 128.3 (×2), 128.2 (×2), 128.1, 127.8,127.6, 100.6, 100.1, 98.8, 98.6, 77.9, 77.4, 76.9, 74.4, 74.2, 72.9,72.8, 72.7, 72.6, 72.5, 72.3, 72.2, 68.1, 67.8, 67.7, 67.5, 67.1, 64.4,64.2, 64.1, 63.1, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6 (×2), 18.4, 18.3ppm; HRMS (ESI): m/z calcd for C₆₆H₇₈N₁₂O₁₆Na [M+Na]⁺: 1317.5551. found:1317.5549.

5′-Methoxycarbonylpentyl4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (79)

Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 78(0.434 g, 0.335 mmol) in CH₃OH (10 mL) until pH˜9 and the resultingmixture was stirred under argon for 4 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.356 g, 89%) as a white foam. Analyticaldata for 79: Rf=0.50 (ethyl acetate/toluene, 1/9, v/v); [α]_(D) ²¹=+65.6(c=1.3, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.13-7.42 (m, 20H, H—Ar),5.10 (d, J_(1,2)=0.9 Hz, 1H, H-1^(D)), 4.93 (d, J_(1,2)=1.7 Hz, 1H,H-1^(C)), 4.90 (d, J_(1,2)=1.8 Hz, 1H, H-1^(B)), 1.19-1.29 ppm (m, 12H,H-6^(A), H-6^(B), H-6^(C), H-6^(D)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0,137.3 (×2), 137.2, 137.1, 128.7, 128.6 (×2), 128.4, 128.3 (×2), 128.2,128.1 (×2), 100.5, 100.2, 98.6, 97.8, 77.8, 77.5, 76.9, 76.5, 74.1,73.1, 72.7, 72.5, 72.3, 72.2, 70.0, 67.9, 67.8, 67.5, 67.3, 67.1, 66.5,64.5, 64.4, 64.3, 51.5, 33.9, 29.1, 25.7, 24.7, 18.7, 18.6, 18.4, 18.3ppm; HRMS (ESI): m/z calcd for C₅₉H₇₄N₁₂O₁₅Na [M+Na]⁺: 1213.5289. found:1213.5284.

5′-Methoxycarbonylpentyl4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (80)

A mixture of glycosyl donor 63 (0.220 g, 0.313 mmol), glycosyl acceptor79 (0.339 g, 0.285 mmol) and freshly activated molecular sieves (3 Å,0.5 g) in CH₂Cl₂ (10 mL) was stirred under argon for 4 h at roomtemperature. MeOTf (260 μL, 2.28 mmol) was added and continued stirringfor additional 48 h. Then Et₃N (1 mL) was added, the solid was filteredoff and the residue was rinsed with CH₂Cl₂ (3×30 mL). The combinedfiltrate (100 mL) was washed with 20% aq. NaHCO₃ (40 mL), water (40 mL),and brine (20 mL). The organic phase was separated, dried over MgSO₄,and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.479 g, 92%) as a white foam. Analyticaldata for 80: Rf=0.60 (ethyl acetate/toluene, 0.5/9.5, v/v); [α]_(D)²¹=+15.3 (c=1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 7.06-8.00 (m, 35H,H—Ar), 5.71 (dd, J_(2,3)=3.3 Hz, 1H, H-2^(F)), 5.58 (dd, J_(3,4)=10.3Hz, 1H, H-3^(F)), 5.09 (d, J_(1,2)=1.2 Hz, 1H, H-1^(E)), 5.05-5.07 (m,2H, H-1^(D), H-1^(F)), 4.95 (d, J_(1,2)=1.5 Hz, 1H, H-1^(C)), 4.90 (d,J_(1,2)=1.7 Hz, 1H, H-1^(B)), 1.20-1.29 ppm (m, 15H, H-6^(A), H-6^(B),H-6^(C), H-6^(D), H-6^(E)); ¹³C NMR (126 MHz, CDCl₃): δ 174.0, 165.2,164.9, 137.5, 137.4, 137.3, 137.2, 137.1, 133.4, 133.3, 129.8 (×2),129.5, 129.4, 129.0, 128.7, 128.6 (×2), 128.5 (×3), 128.4 (×2), 128.3(×2), 128.2 (×2), 128.1, 127.9, 127.8, 127.7, 127.6, 100.9, 100.5,100.2, 99.2, 98.6, 98.2, 77.9, 77.7, 77.5, 76.9, 76.4, 74.1, 73.6, 73.1,72.9, 72.5, 72.4, 72.2 (×2), 72.0, 71.0, 69.4, 68.2, 68.1, 67.9, 67.8,67.7, 67.5, 67.1, 64.7, 64.5, 64.4, 64.3, 63.6, 63.5, 51.5, 33.9, 29.1,25.7, 24.7, 18.6 (×3), 18.5 (×2), 18.4 ppm; HRMS (ESI): m/z calcd forC₉₂H₁₁₀N₁₉O₂₃ [M+NH₄]⁺: 1848.8016. found: 1848.8005.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (77a)

Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 77(0.483 g, 0.264 mmol) in CH₃OH (12 mL) until pH˜9 and the resultingmixture was stirred under argon for 6 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.375 g, 87%) as oil. Analytical data for77a: Rf=0.30 (ethyl acetate/toluene, 1.5/8.5, v/v); [α]_(D) ²¹=+101.4(c=1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.28-7.40 (m, 25H, H—Ar),4.98 (d, J_(1,2)=1.5 Hz, 1H, H-1^(F)), 4.90 (d, J_(1,2)=1.5 Hz, 1H,H-1^(E)), 4.89 (d, J_(1,2)=1.5 Hz, 1H, H-1^(D)), 4.86 (d, J_(1,2)=2.0Hz, 1H, H-1^(C)), 4.85 (d, J_(1,2)=2.0 Hz, 1H, H-1^(B)), 1.13-1.26 ppm(m, 18H, H-6^(A), H-6^(B), H-6^(C), H-6^(D), H-6^(E), H-6^(F)); ¹³C NMR(126 MHz, CDCl₃): δ 174.0, 137.4 (×2), 137.2 (×3), 128.7 (×2), 128.6(×2), 128.4 (×2), 128.3 (×4), 128.1 (×2), 100.7, 100.4, 100.2 (×2),100.1, 98.6, 77.5, 76.9, 76.6 (×2), 74.1, 73.6, 73.5, 73.3, 73.2, 72.3(×2), 72.2 (×2), 70.2, 70.0, 67.9, 67.8 (×2), 67.5, 67.4, 67.1, 65.8,64.4, 64.3, 64.2, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×2), 18.5 (×3),18.2 ppm; HRMS (ESI): m/z calcd for C₇₈H₉₈N₁₈O₂₁Na [M+Na]⁺: 1645.7046.found: 1645.7043.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (48)

To a stirred solution of 77a (0.130 g, 0.080 mmol), in pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., andstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 77b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₇₈H₁₁₁N₆O₂₁ [M+H]+: 1467.7797. found:1467.7795. This crude material was directly used for formylation.

Compound 77b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydride (Angew (1979) Chem. Int. Ed. 18, 614) (5 mL, etherealsolution) and stirred for 3 h, then slowly allowed to warm to roomtemperature. Then solvents were evaporated and the residue was passedthrough column chromatography on silica gel (methanol-dichloromethanegradient elution) to afford disaccharide 77c. HRMS (ESI): m/z calcd forC₈₄H₁₁₁N₆O₂₇Na [M+H]⁺: 1635.7492. found: 1635.7485.

Compound 77c was dissolved in CH₃OH/H₂O (2:1, 5 mL), Pd(OH)₂ on carbon(20%, 0.050 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.052 g, 55%, over 3 steps) as a white foam. Analytical data for 48:Rf=0.20 (CH₃OH/CH₂Cl₂, 2/3, v/v); ¹H NMR (500 MHz, D₂O): δ 8.17-8.19 (Z)and 7.99-8.02 (E) (m, 6H, NCHO), 4.84-5.20 (m, 6H, 6×H-1), 1.16-1.27 ppm(m, 18H, 6×H-6); ¹³C NMR (126 MHz, D₂O): δ178.5, 168.8, 165.9, 103.0,102.9, 101.6, 101.5 (×2), 99.2, 78.6, 78.3, 78.1 (×2), 78.0 (×2), 77.9,69.9 (×2), 69.2 (×2), 69.0, 68.9 (×2), 68.8, 68.7, 68.6 (×2), 68.5 (×3),57.9, 57.7, 53.1, 53.0, 52.9, 52.8 (×2), 52.7, 49.9, 34.6, 29.0, 25.8,24.9, 17.9 (×2), 17.8, 17.7 (×2), 17.6 (×2) ppm; HRMS (ESI): m/z calcdfor C₄₉H₈₀N₆O₂₇Na [M+Na]⁺: 1207.4964. found: 1207.4941.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (85)

A solution of 48 (0.034 g, 0.029 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on a C18 column with a gradient of water-acetonitrile andlyophilized, to give the title compound (0.034 g, 97%) as a white foam.Analytical data for 85: Rf=0.20 (CH₃OH); ¹H NMR (500 MHz, D₂O): δ8.16-8.19 (Z) and 7.98-8.02 (E) (m, 6H, NCHO), 4.81-5.19 (m, 6H, 6×H-1),1.14-1.26 ppm (m, 18H, 6×H-6); ¹³C NMR (126 MHz, D₂O): δ177.4, 177.1,167.9, 167.8, 164.9, 164.6, 102.4, 102.1, 102.0 (×2), 100.8, 100.7,100.6 (×2), 98.6, 98.3 (×2), 77.6, 77.4, 77.3, 77.2, 77.1, 77.0, 76.8,70.4, 70.3, 70.1, 70.0, 69.4, 69.2, 69.0 (×2), 68.3 (×2), 68.0 (×2),67.9 (×2), 67.8 (×2), 67.7 (×2), 67.6 (×2), 67.5 (×2), 67.0, 57.0, 56.9,56.7, 53.9, 53.8, 53.6, 52.0, 51.9 (×2), 51.8 (×2), 51.7, 40.9, 40.4,40.0, 39.7, 35.7, 28.2, 25.0 (×2), 24.9, 17.1 (×2), 17.0 (×2), 16.9(×3), 16.8, 16.7 ppm; HRMS (ESI): m/z calcd for C₅₀H₈₄N₈O₂₆Na [M+Na]⁺:1235.5389. found: 1235.5384.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(91)

To a stirred solution of 85 (0.0142 g, 0.012 mmol) in water (0.5 mL) andEtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20%in ethanol, 55 μL) was added and the pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.014 g,88%) as a white foam. Analytical data for 91: Rf=0.40 (CH₃OH/CH₂Cl₂,1/1, v/v); ¹H NMR (700 MHz, D₂O): δ 8.20-8.23 (Z) and 8.02-8.07 (E) (m,6H, NCHO), 4.88-5.34 (m, 6H, 6×H-1), 1.17-1.35 (m, 20H, —CH_(2d)—,6×H-6), 0.88-1.00 ppm (m, 3H, —CH₃₁); ¹³C NMR (126 MHz, D₂O): δ 189.8,189.6, 184.3, 178.5, 178.1, 177.9, 174.8, 174.7, 168.8, 165.9, 102.9(×2), 101.5 (×3), 99.2 (×2), 78.5, 78.1 (×3), 78.0 (×2), 75.4, 75.3,69.9, 69.2, 69.0, 68.9, 68.7, 68.5, 57.9, 57.7, 53.0, 52.9 (×2), 52.6,45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 32.3, 29.2, 26.1, 26.0, 25.9 (×2),25.9, 19.2, 19.1, 19.0, 17.9 (×2), 17.8 (×2), 17.7 (×2), 13.9 ppm; HRMS(ESI): m/z calcd for C₅₈H₉₂N₈O₂₉Na [M+Na]⁺: 1387.5862. found: 1387.5856.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3)4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2)4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (80a)

Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 80(0.413 g, 0.225 mmol) in CH₃OH (12 mL) until pH˜9 and the resultingmixture was stirred under argon for 6 h at room temperature. Then thereaction mixture was neutralized with Amberlite IR 120 (H⁺) ion exchangeresin, the resin was filtered off and rinsed successively with CH₃OH.The combined filtrate was concentrated in vacuo and purified by columnchromatography on silica gel (ethyl acetate-toluene gradient elution) toafford the title compound (0.334 g, 91%) as a white foam. Analyticaldata for 80a: Rf=0.40 (ethyl acetate/toluene, 1/9, v/v); [α]_(D)²¹=+61.0 (c=1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 7.14-7.38 (m, 25H,H—Ar), 5.03-5.05 (br. s., 2H, H-1^(D), H-1^(E)), 4.94 (d, J_(1,2)=1.5Hz, 1H, H-1^(C)), 4.91 (d, J_(1,2)=1.2 Hz, 1H, H-1^(B)), 4.90 (d,J_(1,2)=1.5 Hz, 1H, H-1^(F)), 1.15-1.35 ppm (m, 20H, —CH_(2d)—, H-6^(A),H-6^(B), H-6^(C), H-6^(D), H-6^(E), H-6^(F)); ¹³C NMR (126 MHz, CDCl₃):δ 174.0, 137.5, 137.4, 137.3, 137.2, 137.1, 128.7, 128.6 (×3), 128.4(×2), 128.3 (×2), 128.2 (×2), 128.1 (×2), 128.0 (×2), 127.9, 127.5,101.0, 100.9, 100.4, 100.2, 98.6, 98.1, 78.0, 77.6, 77.5 (×2), 76.9,76.4, 74.0, 73.3, 73.1, 72.9, 72.5, 72.4, 72.2, 72.1, 71.9, 70.2, 69.9,68.1, 67.9 (×2), 67.8, 67.5 (×2), 67.1, 65.8, 64.6, 64.5, 64.4, 64.3,64.0, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×3), 18.5, 18.4, 18.3 ppm;HRMS (ESI): m/z calcd for C₇₈H₉₈N₁₈O₂₁Na [M+Na]⁺: 1645.7046, found:1645.7035.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl(1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (49)

To a stirred solution of 80a (0.150 g, 0.092 mmol), in pyridine (5 mL)and water (2 mL) mixture, H₂S was bubbled for 0.5 h at 40° C., andstirring was continued for 16 h. Then argon was bubbled through thesolution for 10 min, solvents were removed in vacuo, and the residue wasco-evaporated with toluene (3×10 mL) and dried. The high resolution massspectrometry analysis showed completion of reaction to correspondingamine compound 80b and no products arising from incomplete reduction.HRMS (ESI): m/z calcd for C₇₈H₁₁₁N₆O₂₁ [M+H]+: 1467.7797. found:1467.7781. This crude material was directly used for formylation.

Compound 80b in CH₃OH (5 mL) at −20° C. was added a freshly preparedformic anhydride^([22]) (5 mL, ethereal solution) and stirred for 3 h,then slowly allowed to warm to room temperature. Then solvents wereevaporated and the residue was passed through column chromatography onsilica gel (methanol-dichloromethane gradient elution) to afforddisaccharide 80c. HRMS (ESI): m/z calcd for C₈₄H₁₁₀N₆O₂₇Na [M+Na]⁺:1657.7311. found: 1657.7314.

Compound 80c was dissolved in CH₃OH/H₂O (2:1, 5 mL), Pd(OH)₂ on carbon(20%, 0.050 g) was added. Then it was stirred under a pressure ofhydrogen gas at room temperature for 16 h. After filtration throughcelite pad and washed with CH₃OH (3×10 mL), and solvents were removed invacuo. The residue was purified by column chromatography on silica gel(methanol-dichloromethane gradient elution) to afford the title compound(0.066 g, 60%, over 3 steps) as a white foam. Analytical data for 49:Rf=0.30 (CH₃OH/CH₂Cl₂, 1/1, v/v); ¹H NMR (700 MHz, D₂O): δ 8.20-8.25 (Z)and 8.02-8.06 (E) (m, 6H, NCHO), 4.89-5.23 (m, 6H, 6×H-1), 1.19-1.31 ppm(m, 18H, 6×H-6); ¹³C NMR (176 MHz, D₂O): δ 178.4, 168.6, 165.7 (×2),165.4 (×2), 103.1, 102.4, 101.5 (×2), 101.3, 99.1 (×2), 78.8, 78.4,78.2, 78.0, 77.9, 77.6, 77.2, 69.7 (×2), 69.6 (×2), 69.5, 69.1, 69.0,68.8, 68.7 (×2), 68.6, 68.5, 68.4 (×2), 68.3, 68.2, 67.9, 57.8, 57.7,57.6, 52.9, 52.8, 52.7, 52.6, 52.5, 51.7, 34.4, 28.9, 25.7, 24.8, 18.0,17.9, 17.8 (×2), 17.7 (×2), 17.6 (×3), 17.5 ppm; HRMS (ESI): m/z calcdfor C₄₉H₈₀N₆O₂₇Na [M+Na]⁺: 1207.4964. found: 1207.4963.

(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside (86)

A solution of 49 (0.041 g, 0.035 mmol) in freshly distilled1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excessreagent was removed in vacuo, and the residue was co-evaporated withCH₃OH (3×10 mL) and dried. The residue was purified by reversed phaseHPLC on a C18 column with a gradient of water-acetonitrile andlyophilized, to give the title compound (0.039 g, 93%) as a white foam.Analytical data for 86: R_(f)=0.20 (CH₃OH); ¹H NMR (500 MHz, D₂O): δ8.25-8.32 (Z) and 8.08-8.14 (E) (m, 6H, NCHO), 4.92-5.30 (m, 6H, 6×H-1),1.23-1.39 ppm (m, 18H, 6×H-6); ¹³C NMR (126 MHz, D₂O): δ 178.4, 178.1,168.8 (×2), 165.9, 165.6, 103.3 (×2), 102.6, 101.9, 101.8, 101.6, 101.5,99.3, 99.2, 78.9, 78.6, 78.5, 78.1, 77.8, 77.4, 71.0, 69.9 (×2), 69.7(×2), 69.6, 69.4, 69.2, 69.0, 69.0, 68.9, 68.8, 68.6 (×2), 68.5 (×2),68.4, 68.0, 57.9 (×2), 57.8, 57.7, 56.4, 53.0, 52.8 (×2), 52.7, 51.8,41.4, 41.1, 40.6, 36.6, 29.1, 26.0, 25.9 (×2), 18.1 (×3), 18.0, 17.9,17.8, 17.7, 17.6 ppm; HRMS (ESI): m/z calcd for C₅₀H₈₅N₈O₂₆ [M+H]⁺:1213.5570. found: 1213.5564.

1-[(2′-Aminoethylamido)carbonylpentyl4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2)4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione(92)

To a stirred solution of 86 (0.014 g, 0.011 mmol) in water (0.8 mL) andEtOH (0.6 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20%in ethanol, 55 μL) was added and the pH was adjusted to 8 by carefuladdition of aq. NaHCO₃ (1%) solution. After 0.5 h, TLC showed thereaction was complete; the reaction mixture was neutralized usingCH₃COOH (10%) and concentrated in vacuo. The residue was purified byreversed phase HPLC on a C18 column with a gradient ofwater-acetonitrile and lyophilized, to give the title compound (0.012 g,76%) as a white foam. Analytical data for 92: Rf=0.40 (CH₃OH/CH₂Cl₂,1/1, v/v); ¹H NMR (700 MHz, D₂O): δ 8.20-8.25 (Z) and 8.02-8.07 (E) (m,6H, NCHO), 4.85-5.21 (m, 6H, 6×H-1), 1.18-1.35 (m, 20H, —CH_(2d)—,6×H-6), 0.89-0.97 ppm (m, 3H, —CH₃₁); ¹³C NMR (126 MHz, D₂O): δ 189.8,189.7, 184.3, 178.5, 178.1, 177.9 (×2), 174.8, 174.7, 168.8, 165.9,165.6, 103.3, 102.6, 101.6, 101.5, 99.2, 78.9, 78.5, 78.2, 77.8, 77.3,75.4, 75.3, 70.8, 69.9, 69.7, 69.2, 69.0, 68.8, 68.7, 68.6, 68.5, 68.4,57.9, 57.7, 53.0, 52.8, 52.7, 51.8, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4,29.2, 26.1, 26.0, 25.9 (×2), 19.1, 19.0, 18.1, 18.0, 17.9 (×2), 17.8,17.7, 13.9 ppm; HRMS (ESI): m/z calcd for C₅₈H₉₂N₈O₂₉Na [M+Na]⁺:1387.5862. found: 1387.5864.

Conjugation of the Pentasaccharide Squarate 40 to BSA (42)

BSA (11.7 mg, 0.17 μmol) and squarate 40 (4.2 mg, 3.5 μmol) weredissolved in 0.5 M borate buffer pH 9 (350 μL) and stirred gently atroom temperature for 3 days. Then the reaction mixture was diluted withwater and dialyzed against deionized water (3×2 L) at 4° C., spinned andlyophilized. The product of conjugation was obtained as a white solid(12.5 mg, 84%): MALDI-TOF-MS indicated the conjugate 42 had an averageof 16.4 pentasaccharides per BSA.

Conjugation of the Nonasaccharide Squarate 41 to BSA (43)

The conjugation of BSA (5.5 mg, 0.08 μmol) and squarate 41 (3.2 mg, 1.6μmol) in borate buffer (220 μl) was performed as described for 42. Afterthe dialysis (5×2 L) and lyophilization the conjugate 43 was obtained asa white solid (6 mg, 74%). As calculated from MALDI-TOF-MS spectrum theaverage number of nonasaccharides per BSA was 16.8.

Conjugation of Squarate Derivative 87 to BSA (93).

BSA (30 mg, 0.451 μmol) and disaccharide squarate 87 (4.5 mg, 6.77 μmol)were dissolved in 0.5 M borate buffer pH 9 (600 μL) and stirred gentlyat room temperature for 3 days. Then the reaction mixture was dilutedwith Mili-Q water, filtered through milipore filtration tube (10,000MWCO, 4×10 mL), lyophilized and the BSA-conjugate 93 was obtained as awhite foam (30.4 mg, 89%). The MALDI-TOF mass spectrometry analysisindicated the conjugate 93 had an average of 15.2 disaccharides per BSA.

Conjugation of Squarate Derivative 88 to BSA (94).

BSA (30 mg, 0.451 μmol) and trisaccharide squarate 88 (5.7 mg, 6.74μmol) were dissolved in 0.5 M borate buffer pH 9 (700 μL) and stirredgently at room temperature for 3 days. Then the reaction mixture wasdiluted with Mili-Q water, filtered through milipore filtration tube(10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 94 wasobtained as a white foam (32.3 mg, 91%). The MALDI-TOF mass spectrometryanalysis indicated the conjugate 94 had an average of 15.9trisaccharides per BSA.

Conjugation of Squarate Derivative 89 to BSA (95).

BSA (32.5 mg, 0.489 μmol) and trisaccharide squarate 89 (6.2 mg, 7.34μmol) were dissolved in 0.5 M borate buffer pH 9 (700 μL) and stirredgently at room temperature for 3 days. Then the reaction mixture wasdiluted with Mili-Q water, filtered through milipore filtration tube(10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 95 wasobtained as a white foam (33.5 mg, 87%). The MALDI-TOF mass spectrometryanalysis indicated the conjugate 95 had an average of 15.7trisaccharides per BSA.

Conjugation of Squarate Derivative 90 to BSA (96).

BSA (11 mg, 0.165 μmol) and tetrasaccharide squarate 90 (2.5 mg, 2.45μmol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirredgently at room temperature for 3 days. Then the reaction mixture wasdiluted with Mili-Q water, filtered through milipore filtration tube(10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 96 wasobtained as a white foam (12 mg, 92%). The MALDI-TOF mass spectrometryanalysis indicated the conjugate 96 had an average of 13.4tetrasaccharides per BSA.

Conjugation of Squarate Derivative 91 to BSA (97).

BSA (5 mg, 0.0752 μmol) and hexasaccharide squarate 91 (1.5 mg, 1.099μmol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirredgently at room temperature for 3 days. Then the reaction mixture wasdiluted with Mili-Q water, filtered through milipore filtration tube(10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 97 wasobtained as a white foam (5.5 mg, 87%). The MALDI-TOF mass spectrometryanalysis indicated the conjugate 97 had an average of 13.8hexasaccharides per BSA.

Conjugation of Squarate Derivative 92 to BSA (98).

BSA (5 mg, 0.0752 μmol) and hexasaccharide squarate 92 (1.5 mg, 1.099μmol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirredgently at room temperature for 2 days. Then the reaction mixture wasdiluted with Mili-Q water, filtered through milipore filtration tube(10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 98 wasobtained as a white foam (6 mg, 99%). The MALDI-TOF mass spectrometryanalysis indicated the conjugate 98 had an average of 10.8hexasaccharides per BSA.

Conjugation of Squarate Derivative 87 to Co Povidone Polymer (99).

High loading: Co-povidone (6.9 mg, 5.95 μmol, 1 eq.) and disaccharidesquarate 87 (2.0 mg, 2.97 μmol, 0.5 eq.) were dissolved in 0.5 M boratebuffer pH 9 (500 μL) and stirred gently at room temperature for 2 days.Then, a solution of 5% aq. Ac₂O (1 mL) and saturated NaHCO₃ (1 mL) wasadded and stirred for 3 h. After that, the reaction mixture was dilutedwith Mili-Q water, dialyzed against deionized water (5×2 L), lyophilizedto obtain the co-povidone-conjugate 99a.

Low Loading:

Co-povidone (5.0 mg, 4.31 μmol, 1 eq.) and disaccharide squarate 87 (0.5mg, 0.74 μmol, 0.166 eq.) were dissolved in 0.5 M borate buffer pH 9(500 μL) and stirred gently at room temperature for 2 days. Then, asolution of 5% aq. Ac₂O (1 mL) and saturated NaHCO₃ (1 mL) was added andstirred for 3 h. After that, the reaction mixture was diluted withMili-Q water, dialyzed against deionized water (5×2 L), lyophilized toobtain the co-povidone-conjugate 99b.

Conjugation of Squarate Derivative 91 to Co-Povidone Polymer (100). HighLoading:

Co-povidone (5.0 mg, 4.31 μmol, 1 eq.) and hexasaccharide squarate 91(2.94 mg, 2.15 μmol, 0.5 eq.) were dissolved in 0.5 M borate buffer pH 9(500 μL) and stirred gently at room temperature for 2 days. Then, asolution of 5% aq. Ac₂O (1 mL) and saturated NaHCO₃ (1 mL) was added andstirred for 3 h. After that, the reaction mixture was diluted withMili-Q water, dialyzed against deionized water (5×2 L), lyophilized toobtain the co-povidone-conjugate 100a.

Low Loading:

Co-povidone (5.0 mg, 4.31 μmol, 1 eq.) and hexasaccharide squarate 91(1.0 mg, 0.73 μmol, 0.166 eq.) were dissolved in 0.5 M borate buffer pH9 (500 μL) and stirred gently at room temperature for 2 days. Then, asolution of 5% aq. Ac₂O (1 mL) and saturated NaHCO₃ (1 mL) was added andstirred for 3 h. After that, the reaction mixture was diluted withMili-Q water, dialyzed against deionized water (5×2 L), lyophilized toobtain the co-povidone-conjugate 100b.

Conjugation of Squarate Derivative 91 to Tetanus Toxoid (101).

Hexasaccharide squarate 91 (0.55 mg, 0.403 μmol) was added to thesolution of tetanus toxoid (2 mg, 0.0133 μmol) in 0.5 M borate buffer pH9 (1 mL) and stirred gently at room temperature for 3 days. Then thereaction mixture was washed with borate buffer, filtered throughmilipore filtration tube (10,000 MWCO, 4×10 mL) and the resultingtetanus toxoid-conjugate 101 was stored in PBS buffer. The MALDI-TOFmass spectrometry analysis indicated the conjugate 101 had an average of12.6 hexasaccharides per tetanus toxoid.

Conjugation of Squarate Derivative 92 to Tetanus Toxoid (102).

Hexasaccharide squarate 92 (0.55 mg, 0.403 μmol) was added to thesolution of tetanus toxoid (2 mg, 0.0133 μmol) in 0.5 M borate buffer pH9 (1 mL) and stirred gently at room temperature for 2 days. Then thereaction mixture was washed with borate buffer, filtered throughmilipore filtration tube (10,000 MWCO, 4×10 mL) and the resultingtetanus toxoid-conjugate 102 was stored in PBS buffer. The MALDI-TOFmass spectrometry analysis indicated the conjugate 102 had an average of6.2 hexasaccharides per tetanus toxoid.

Specific M-Antigen—Proof of Concept Studies on OPS from Brucella and Y.enterocolitica O:9

Monoclonal antibody binding analysis using the anti-M specific antibodyBM40 (Greiser et al. (1987) Ann Inst Pasteur Microbiol 138, 549-560)against Brucella A, M and Y. enterocolitica O:9 sLPS antigens, preparedby hot-phenol extraction (Westphal et al. (1952) Z. Naturforsch. 7,148-155) confirmed the exceptionally high specificity of the BM40response in this case. There was no measurable binding against the Y.enterocolitica O:9 sLPS and binding to B. melitensis 16M (an M dominantstrain) was tenfold greater than binding to B. abortus S99 (an Adominant strain).

During further exploratory studies, the absorption methods describedabove (Alton et al. (1994) Techniques for the Brucellosis Laboratory,pages 53-54; INRA Editions, ISBN-10: 2738000428; Kittelberger et al.(1998) Vet. Microbial. 60, 45-57) were evaluated, using sera from cattleexperimentally infected with Brucella abortus strain 544 (an A dominantstrain) or Y. enterocolitica O:9. A residual anti-Brucella sLPS titrewas observed in indirect ELISA in Y. enterocolitica O:9 absorbed samplesin some, but not all, sera from the Brucella infected animals.

The sera from the experimentally infected cattle was also evaluated byindirect ELISAs using B. melitensis 16M and Y. enterocolitica O:9 OPSantigens purified from the smooth LPS using mild acid hydrolysis andsize exclusion chromatography (Meikle et al. (1989) Infect Immun 57,2820-2828), tested to be free of lipid-A using the limulus amebocytelysate reaction (Ding et al. (2001) Trends Biotechnol. 19, 277-281).Samples taken from the cattle (cows) 3, 7, 16, 24 and 53 weeks postinfection were taken from each of the four animals experimentallyinfected with B. abortus strain 544 and from each of the four animalsexperimentally infected with Y. enterocolitica O:9. These samples havebeen previously described and also tested by conventional serologicalassays for brucellosis as described previously (McGiven et al. (2008)Journal of Immunological Methods 20, 7-15).

The OPS from B. melitensis 16M was chosen in preference to OPS from a B.abortus A dominant strain due to the higher frequency of α-1,3 linkagesand M epitopes, so that the extremes of the structural variation in thenatural 4,6-dideoxy-4-formamido-α-D-mannopyranose homopolymers would berepresented.

Owing to their high solubility, the purified OPS antigens do not bindeffectively to ELISA polystyrene ELISA plates by passive absorption(unless conjugated to carrier molecules containing hydrophobic regions).To enable their immobilisation to a solid phase, Carbo-BIND™ 96 wellELISA plates were used (Corning, product number 2507) which havehydrazide functional groups on the well surface. These hydrazide groupsreact spontaneously with aldehydes which may be generated incarbohydrates by oxidation.

The OPS was oxidised at 100 μg/ml with 10 mM sodium metaperiodate and 50mM sodium acetate buffer (SAB) pH 5.5 in the dark at 4° C. for 30minutes. After this time the antigen was diluted to between 0.5 to 0.125μg/ml in SAB pH 5.5 and 100 μl was added to the wells of a Carbo-BIND™plate. The plate was incubated for 1 hour at 37° C. and then washed with4 times 200 μl of phosphate buffered saline with 0.05% Tween-20(PBS-T20) and tapped dry.

Serum was diluted 1/50 in buffer (Sigma #B6429) and 100 μl of this wasadded per well of the antigen coated plate, each sample tested induplicate. The plate was incubated for 1 hr at room temperature on arotary shaker at 160 rpm and then washed with PBS-T20 as describedabove.

An HRP conjugated Protein-G conjugate (Thermo #31499), diluted to 1μg/ml in buffer, was then added in 100 μl volumes to each well of theplate which was then incubated and washed as described above for serum.The plate was then developed with ABTS(2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mMsodium azide and read at 405 nm wavelength. The optical density for theduplicates was averaged and the blank OD (buffer only instead of sera)was subtracted. This value was then expressed as a percentage of acommon positive control serum sample from a B. abortus biovar 1 (Adominant) infected bovine for cattle sera.

As expected, there was much cross reaction between the Brucella and Y.enterocolitica O:9 OPS antigens with both sets of sera, i.e., those fromthe cattle experimentally infected with Brucella abortus strain 544 andthose experimentally infected with Y. enterocolitica O:9. An additionaloutput was calculated for each sample by dividing the result from the B.melitensis OPS iELISA by that from the Y. enterocolitica O:9 OPS iELISAto derive a simple ratiometric value (henceforth described as the‘Bm16M/YeO:9 Ratio’). Thus, higher Bm16M/YeO:9 Ratio values signify thatthe intensity of the reaction to the B. melitensis 16M OPS antigen wasgreater than the intensity of the reaction to the Y. enterocolitica O:9antigen relative to lower Bm16M/YeO:9 Ratio values. Analysis of thisdata set revealed that there was a highly significant difference(P=0.008, Student's t-test for unpaired data) between the theBm16M/YeO:9 Ratio values for the two different sample populations,whereby the mean value from the Brucella infected animals was higherthan the mean value from the Y. enterocolitica O:9 infected animals.There was, therefore, a significant difference in the way that the serumfrom the two populations reacted to these two antigen types, with thesera from the Brucella infected animals reacting with a relativelyhigher intensity to the B. melitensis 16M OPS and vice versa. Thisresult suggests that there are significant and detectable differences inthe anti-OPS antibody repertoire between the two serum populations.

As a consequence of this finding with the relative OPS ELISA results,the same methods were applied to archived bovine field sera from GreatBritain. A total of 45 samples from individual animals confirmed byculture to be infected with B. abortus biovar 1 (A dominant) weretested. Also 68 samples were tested from individual animals whose serawas collected from 1996 to 1999, more than 10 years since thedeclaration of officially brucellosis free status for Great Britain,that were positive in conventional serology for brucellosis such as B.abortus S99 sLPS iELISA, SAT and CFT (Nielsen et al. (2009) “Bovinebrucellosis” In: Manual of Diagnostic Tests & Vaccines for TerrestrialAnimals 2009; Office International Des Epizooties, Paris, pg 10-19) butfor which there was no cultural or epidemiological evidence ofbrucellosis. As with the data from the experimentally infected animalsthere was a highly significant difference (P=0.000012, Student's t-testfor unpaired data) between the Bm16/YeO:9 Ratio values for the twodifferent serological groups. As before, sera from cattle with confirmedbrucellosis had, on average, higher values (FIG. 2).

The results from both the B. melitensis and Y. enterocolitica O:9 OPSiELISAs were evaluated to find the positive/negative cut-off for eachwhich generated the highest Youden Index (YI=diagnostic sensitivity[DSn]+diagnostic specificity [DSp]−1). These optimised YI values withthe associate DSn and DSp figures are shown in Table 3.

TABLE 3 Performance statistics for OPS iELISAs as tested on bovine seraOptimal Youden Index (YI = DSn + DSp-1) ROC - Area Under Curve YI DSnDSp 95% Confidence Assay Estimate % % AUC Interval Y. enterocolitica0.1297 64.44 48.53 0.5065 0.3964-0.6167 O:9 OPS iELISA B. melitensis0.4232 55.56 86.76 0.7065 0.6024-0.8106 16M OPS iELISA Bm16M/YeO:90.5343 66.67 86.76 0.8056 0.7219-0.8892 Ratio

To further evaluate the diagnostic effectiveness of each of the OPSiELISAs Receiver Operator Characteristic (ROC) Curve analysis was used,in particular the evaluation of the Area Under the Curve (AUC) (Hanleyand McNeil (1982) Radiology 143, 29-36). In this context, the AUCrepresents the ability of the assay to correctly classify samples fromanimals that are Brucella infected and those that are not. The data inTable 2 shows that the B. melitensis OPS iELISA has a higher optimisedYI and AUC value than the Y. enterocolitica O:9 OPS iELISA.

In Table 4, P values relating to testing for significant differencesbetween AUC data are presented. Testing for the significance ofdifferences between AUC values was performed using the method for pairedsamples (Hanley and McNeil (1983) Radiology 148, 839-843).

This data shows that there is a highly significant difference (P<0.0001)between the AUC values for the Y. enterocolitica O:9 and B. melitensis16M OPS iELISAs and therefore the assay is significantly superior. Theimprovement in diagnostic performance when using the Bm16M/YeO:9 Ratiovalues is also demonstrated in Tables 3 and 4. The optimal YI value isgreater than that for both of the individual OPS iELISA assays. The DSpis equal to that of the B. melitensis 16M OPS iELISA and the DSn issuperior. The AUC is also greater, but not significantly so, compared tothe AUC for the B. melitensis 16m OPS iELISA (P=0.239).

This is strong evidence to demonstrate that a combinational ratiometricapproach to the determination of infection status can be moreadvantageous than the interpretation of one test alone when there issignificant cross reaction due to similar but non-identical antigens.The data for the B. melitensis 16M OPS iELISA alone and in combinationas a ratiometric assay suggests that the M epitope is playing a majorrole in the significant differences in antibody binding that have beenobserved. The Bm16M/YeO:9 Ratio evaluation of the samples is arelatively crude attempt to delineate the contribution to overall titremade by the specific antibodies and epitopes from the contribution madeby the common ones.

The same OPS iELISA methods were also applied to 41 samples fromindividual swine that were positive to the Rose Bengal Test (RBT) andiELISA (Olsen, (2010) “Porcine Brucellosis” In: Office International DesEpizooties, Paris) and from herds confirmed by culture to be infectedwith B. suis biovar 1, an A dominant OPS biovar (Meikle et al. (1989)Infect Immun 57, 2820-2828; Olsen, (2009) “Porcine Brucellosis” In:Office International Des Epizooties, Paris, pages 3-4). A further 52samples were tested which were collected from individual animals inGreat Britain, officially free of B. suis, within herds which from whichone or more sample positive in conventional serology such as RBT,cELISA, iELISA (Olsen, (2009) “Porcine Brucellosis” In: OfficeInternational Des Epizooties, Paris, pages 3-4) where obtained and wherethere was no epidemiological evidence of brucellosis.

As with the data from the cattle sera samples, there was a highlysignificant difference (P=0.000000006, using the unpaired Student'st-test) between the Bm16/YeO:9 Ratio values for the two differentserological groups. As before, sera from swine with confirmedbrucellosis had a higher, on average, values than did the false positiveserological samples (FIG. 3A). These samples were also tested by B.abortus S99 (A dominant) OPS iELISA (FIG. 3B) and a ratiometricexpression of the data, analogous to the Bm16M/YeO:9 Ratio, wasevaluated. There was a significant difference between the ratio of theB. abortus S99 to Y. enterocolitica O:9 OPS iELISA results for thedifferent serum groups, but the difference was not as strong (P=0.023)as observed with the Bm16M/YeO:9 Ratio. In fact, the difference betweenthe results from the two Brucella OPS antigens, B. melitensis 16M and B.abortus S99, was much more significant (P=0.000000016) than thedifference between the Y. enterocolitica O:9 and B. abortus S99 results.This reflects the known structure of the these antigens with the Adominant OPS of B. abortus S99 having only approximately 2% α-1,3linkages compared to the M dominant B. melitensis 16M OPS with 20% andY. enterocolitica with 0%.

The optimised YI, as described above, for the individual OPS iELISAs andthe Bm16M/YeO:9 Ratio, results for the swine sera are shown in Table 5where: Bm16M/YeO:9>B. melitensis 16M>B. abortus S99>Y. enterocoliticaO:9.

TABLE 5 Performance statistics for OPS iELISAs as tested on porcine seraOptimal Youden Index (YI = DSn + DSp-1) ROC - Area Under Curve YI DSnDSp 95% Confidence Assay Estimate % % AUC Interval Y. enterocolitica0.3588 87.80 48.08 0.6445 0.5328-0.7562 O:9 OPS iELISA B. abortus S990.6472 87.80 76.92 0.8607 0.7832-0.9382 OPS iELISA B. melitensis 0.708780.49 90.38 0.9135 0.8559-0.9710 16M OPS iELISA B. melitensis 0.767890.24 86.54 0.9085 0.8437-0.9733 16M/ Y. enterocolitica O:9

The data for the AUC was similar whereby: B. melitensis 16M>Bm16M/YeO:9Ratio>B. abortus S99>Y. enterocolitica O:9. The AUC for the Y.enterocolitica O:9 OPS iELISA was significantly lower (P<0.0001) thanfor the other three outputs (Table 6). There was a difference of weaksignificance between the AUC for the B. abortus and B. melitensis OPSiELISAs (P=0.066).

The same methods were applied to 21 serum samples from individual ELISApositive swine from herds confirmed by culture to be infected with B.suis biovar 2 and compared to the data from the 52 samples fromnon-infected swine, but seropositive herds, in Great Britain, as well asto the 41 samples from B. suis biovar 1 infected animals. There was nosignificant difference in the Bm16M/YeO:9 Ratio results between the serafrom the B. suis biovar 2 infected animals and from sera from thenon-infected animals (P=0.926). This is in keeping with the recentdiscovery that, uniquely for Brucella, the OPS from B suis biovar 2contains no α-1,3 linkages (Zaccheus et al. (2013) PLoS One 8, e53941)and therefore the OPS is highly similar, if not identical, to that of Y.enterocolitica O:9.

The data from the swine sera demonstrates that assays using OPS withhigher proportions of α-1,3 linkages provide superior diagnosticattributes. This is further evidence that the α-1,3 linkage is part of asignificant and discriminating epitope for polyclonal sera derived fromanimals naturally infected with Brucella.

Developing the Discrete M Epitope Antigen (Specific M-Antigen)

The evidence described above from the OPS iELISAs provided the stimulusfor additional studies to define, isolate and apply the ‘M’ epitope tothe serodiagnosis of brucellosis in order to increase the accuracy ofthe results. The purified native OPS from Y. enterocolitica O:9, B.melitensis 16M (‘M’ dominant) and B. abortus S99 (‘A’ dominant) waspartially hydrolysed using hot concentrated hydrochloric acid to obtaindi- to dodeca-saccharides. These were evaluated by LC-ESI-MS/MS using agraphitized carbon column (Ruhaak et al. (2009) Anal. Bioanal. Chem.394, 163-174) to separate them and confirm their identity as4,6-dideoxy-4-formamido-α-D-mannopyranosyl oligomers.

The oligosaccharides from the B. abortus S99 OPS were subjected toaffinity chromatography using an affinity chromatography column withimmobilised Brucella anti-M monoclonal antibody BM40 and the wash andelution fractions were further evaluated by LC-ESI-MS/MS using agraphitized carbon column (Ruhaak et al. (2009) Anal. Bioanal. Chem.394, 163-174) to separate the oligosaccharides. Tetrasaccharides of4,6-dideoxy-4-formamido-α-D-mannopyranosyl were readily detectable inthe bound and subsequently eluted, using dilute hydrochloric acid,fractions; the LC-ESI-MS chromatogram was compared to those for thetetrasaccharides found in the non-affinity selected oligosaccharidesderived from the partial acid hydrolysis of the three native antigens.

Whereas the chromatogram for the B. melitensis 16M derivedtetrasaccharide was relatively simple (FIG. 4A), those for B. abortusS99 (FIG. 4B) and Y. enterocolitica O:9 (FIG. 4C) were more complex. Asthe mass was the same across the chromatogram, the differences inelution profile were most likely due to changes in conformation andinteraction with other oligosaccharides during separation within thegraphitised column. What is evident is that the major peak found in theB. melitensis 16M tetrasaccharide chromatograph, eluting at about 9mins, is also found in the B. abortus S99 but not the Y. enterocoliticaO:9 chromatograph. The chromatograph of the B. abortus S99tetrasaccharide affinity selected by the anti-M monoclonal antibody(FIG. 4D) looks extremely similar to the unselected tetrasaccharide fromthe acid hydrolysed native B. melitensis 16M OPS preparation. Theconclusion from this study is that a tetrasaccharide from the BrucellaOPS is large enough to form a viable antibody epitope and that theBrucella specific anti-M monoclonal antibody binds to a tetrasaccharidethat is detectable in B. abortus and not in Y. enterocolitica O:9. Fromthe pre-existing structural knowledge, the only known difference is theα-1,3 linkage.

All the evidence presented above provided a good basis for thehypothesis that a tetrasaccharide antigen containing the M epitope,whilst minimising any C/Y or A epitope-like properties, would make auseful serodiagnostic antigen. Four4,6-dideoxy-4-formamido-α-D-mannopyranose residues were synthesised(Sussex Research Laboratories Inc, Ottawa, Ontario, Canada) within atetrasaccharide that is sequentially α-1,2, α-1,3 and α-1,2 linked,generating a tetrasaccharide having Formula VIII:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (Formula VIII)

This molecule had not previously been synthesised and applied and isreferred to below as the TSM antigen (the “tetrasaccharide M [like]antigen”). The key aspect of this structure is the inclusion of theα-1,3 linkage whilst incorporating the minimal number of α-1,2 linkagesthought necessary for M specific antibodies to bind; this has the effectof minimising the binding of non-M specific antibodies.

Analysis of the TSM Antigen

The structure of the antigen was confirmed by MALDI-ToF and ESI-QToFmass spectrometry. The monoclonal binding properties were confirmed bycompetitive ELISA whereby a standard polystyrene ELISA plate was coatedwith B. melitensis 16M sLPS antigen by passive absorption in carbonatebuffer. The plate was co-incubated with the anti-M monoclonal antibodyBM40 and native OPS from B. melitensis 16M, B. abortus S99, Y.enterocolitica O:9, the TSM antigen as well as buffer controls. Afterwashing the plate, an HRP conjugate specific for the BM40 was added andincubated. After washing, the plates were developed with an HRPsubstrate to determine the degree to which mAb-conjugate binding hadbeen inhibited.

The results (FIG. 5) demonstrated that the TSM antigen was able toinhibit the binding to a greater extent than the equivalentconcentration of Y. enterocolitica O:9 OPS and to a similar degree asthe B. abortus S99 OPS. Taking into account the monovalency of the TSMantigen compared to the multiple binding sites available on 16M OPS, theresults provide strong evidence for specific TSM-antibody binding.

A similar competitive ELISA was conducted to measure the inhibition ofbinding of anti-A and anti-M rabbit hyperimmunised and absorbedmonospecific sera (sera as described previously) using native OPS fromB. melitensis 16M, B. abortus S99, Y. enterocolitica O:9, the TSMantigen as well as buffer controls. The results demonstratedpreferential inhibition of the anti-M sera by the TSM (FIG. 6A) comparedto the inhibition of the anti-A sera (FIG. 6B), in agreement with thetheoretical expectations.

Application of the TSM Antigen to Serodiagnosis

The TSM antigen was then chemically conjugated to an ELISA plate tocreate an indirect ELISA and enable the evaluation of non-competitiveantibody interactions.

Owing to the highly soluble nature of the antigen and its small size,passive absorption to an ELISA plate surface was not straightforward.Instead, it was chemically conjugated to an ELISA plate surfacefunctionalised with hydrazide groups on the end of spacer arms (CorningCarbo-BIND™ #2507). As described above, the hydrazide groups reactspontaneously with aldehydes that may be created within carbohydrates byperiodate oxidation.

The TSM antigen was incubated in 2 mM sodium metaperiodate, 50 mM sodiumacetate buffer at pH 5.5 for 2.5 hours in the dark at 4° C. This wassufficient to oxidise the vicinal diol hydroxyl groups on the 2^(nd) and3^(rd) carbons of the terminal sugar (Sussich et al. (2000) CarbohydrRes 329, 87-95), the only vicinal diol group within the molecule, togenerate the structure shown below:

Competitive ELISA with BM40 anti-M monoclonal antibody was applied asdescribed above to confirm that the oxidised TSM antigen (oxiTSM) shownabove maintained its antibody interaction. Residual sodium metaperiodatewas removed from the reaction by filtration of the TSM antigen andbuffer through a short column of Sephadex G-10 (GE Healthcare) whichreacted with the excess sodium metaperiodate and, with a suitable volumeof elution buffer, allowed the oxiTSM antigen to flow through. Removalof the sodium metaperiodate was necessary to prevent any furtheroxidation of the oxiTSM and removal was confirmed by abrogation of thereaction with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt).

The oxiTSM antigen was then diluted to 10 μg/ml in 0.1 M sodium acetatebuffer pH 5.5 and 100 μl was added per well to a Carbo-BIND™ plate whichwas incubated at 37° C. for 3 hrs. During this incubation process thealdehyde groups on the oxidised tetrasaccharide spontaneously reactedwith the hydrazide groups on the end of a linker attached to the ELISAplate surface to form stable covalent hydrazone bonds, as shown below(the hydrazone bond is shown with a dotted circle):

After this time the plate was washed with 4 times 200 μl per well ofphosphate buffered saline with 0.5% Tween-20 (PBS-T20) and tapped dry.These coated ELISA plates were used to test the field sera from cattle(described above) confirmed by culture to be infected with B. abortus(n=45) and from cattle without brucellosis but with serum cross reactingin one or more conventional serodiagnostic assay (n=68).

Serum was diluted 1/50 in buffer (Sigma #B6429) and 100 μl of this wasadded per well of the antigen coated plate, each sample tested induplicate. The plate was incubated for 1 hr at room temperature on arotary shaker at 160 rpm and then washed with PBS-T20 as describedabove. An HRP conjugated Protein-G conjugate (Thermo #31499), diluted to1 μg/ml in buffer, was then added in 100 μl volumes to each well of theplate which is then incubated and washed as described above for serum.The plate was then developed with ABTS(2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mMsodium azide and read at 405 nm wavelength. The optical density for theduplicates was averaged and the blank OD (buffer only instead of sera)was subtracted. This value was then expressed as a percentage of acommon positive control serum sample from a Brucella infected bovine.

The TSM antigen iELISA was evaluated against the same population ofbovine samples as described above for the evaluation of the B.melitensis 16M and Y. enterocolitica O:9 OPS iELISAs: 45 from B. abortusbiovar 1 (A dominant) culture positive animals and 68 from falsepositive serological reactors. The results for the ELISA are shown inFIG. 7.

Further Antigen Oligosaccharide Conjugates—ELISA Titrations

To begin to determine the overall length of the oligosaccharidenecessary to provide specificity for binding to anti-A or anti-Mantibodies, synthetic pentasaccharide and nonasaccharide BSA conjugateantigens were evaluated against the bovine sera described above. Eachantigen contains one internally and centrally positioned α-1,3 link withthe remaining links being α-1,2.

The pentasaccharide had Formula XI:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (Formula XI)

The nonasaccharide had Formula XV:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (Formula XV)

In Formulae XI and XV above, the central4,6-dideoxy-4-formamido-D-mannopyranose is underlined. The antigens wereconjugated to BSA via a reducing end -1-O—(CH₂)₅—COO—CH₃ linker asdescribed above, to form structures 42 and 43, respectively, shownbelow.

In one experiment, BSA-carbohydrate-protein conjugates 42 and 43 (5μg/mL in PBS) were used to coat 96-well microtiter plates (MaxiSorp,Nunc) overnight at 4° C. The plate was washed 5 times with PBST (PBScontaining 0.05% (v/v) Tween 20). Serial √10 dilutions of mAb YsT9.1 andBm10 ascites fluids or supernatants form hybridoma cell culture weremade in PBST containing 0.1% BSA. The solutions were distributed induplicate on the coated microtiter plate and incubated at roomtemperature for 2 hours. The plate was washed with PBST (5 times) andgoat anti-mouse IgG antibody conjugated to horseradish peroxidase(Kirkegaard & Perry Laboratories; 1:2000 dilution in 0.1% BSA/PBST; 100μL/well) was added. The mixture was then incubated for 1 hour. The platewas washed 5 times with PBST before addition of a 1:1 mixture3,3′,5,5′-tetramethylbenzidine (0.4 g/L) and 0.02% H₂O₂ solution(Kirkegaard & Perry Laboratories; 100 μL/well). After 2 minutes, thereaction was stopped by addition of 1 M phosphoric acid (100 μL/well).Absorbance was read at 450 nm. End point titres are recorded as thedilution giving an absorbance 0.2 above background.

The monoclonal antibodies (YsT9-1 and BM10) previously shown to bespecific for the Brucella A and M antigens (Bundle et al. (1989) Infect.Immun. 57, 2829-2836). were titred to their end point against theantigens 42 and 43 coated on ELISA plates (FIG. 8). The nonasaccharideantigen 43 binds anti-A and M specific antibodies with equivalentavidity, whereas the pentasaccharide 42 displays a preference for the Mspecific antibody, while still binding the A specific antibody but withan approximately 10 fold reduced avidity.

Previous studies showed the YsT9-1 and Bm10 antibodies possessed aviditydifferences of between 400-1,000 for the respective O-polysaccharideantigens (Bundle et al. (1989). Infect. Immun. 57, 2829-2836). Asmentioned, the pentasaccharide antigen 42 shows a preference for Mspecific antibody.

Further Antigen-Oligosaccharide Conjugates—Serology Studies and Analysis

In a further experiment, the conjugates 42 and 43 were immobilised ontothe surface of standard polystyrene ELISA plates passively via overnightincubation in carbonate buffer at 4° C. at 2.5 μg/ml, 100 μl/well. Theplates were washed as described above and incubated with a 1/50 dilutionof sera in buffer (in duplicate)(see below) for 30 mins at roomtemperature at 160 rpm, after which time they were washed and tapped dryas described above. For bovine sera, an HRP-conjugated mouse anti-bovineIgG1 conjugate was used. The conjugates were diluted to working strengthin buffer and the plates incubated, washed and tapped dry as for theserum incubation stage. The plate was then developed with ABTS(2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mMsodium azide and read at 405 nm wavelength. The optical density for theduplicates was averaged and the blank OD (buffer only instead of sera)was subtracted. This value was then expressed as a percentage of acommon positive control serum sample from a Brucella infected bovine.

The same method employed to evaluate bovine serum by iELISA with thepentasaccharide and nonasaccharide conjugates was used to evaluatebovine serum by a further four oligosaccharide BSA conjugates. Theoligosaccharides were:

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (tetrasaccharide, Formula VIII)

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl  (trisaccharide (terminal α-1,3 link),Formula XVI)

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl  (trisaccharide (terminal α-1,2 link),Formula XVII)

4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranose  (disaccharide,Formula II)

These antigens were conjugated to BSA via a reducing end-1-O—(CH₂)₅—COO—CH₃ linker as described above, to form structuressimilar to 42 and 43 shown above. Since conjugation occurs via thereducing end, the link at the non-reducing end is referred to as the“terminal link”.

The BSA-nonasaccharide, pentasaccharide, tetrasaccharide, trisaccharideand disaccharide conjugate iELISAs (and the oxiTSM iELISA describedabove) were evaluated against the same population of bovine fieldsamples as described above for the evaluation of the B. melitensis 16Mand Y. enterocolitica O:9 OPS iELISAs; that is, 45 from B. abortusbiovar 1 (A dominant) culture positive animals and 68 from falsepositive serological reactors. The results for these iELISAs, shown byscatter plot in FIGS. 7 and 9 to 11, were evaluated to find the optimalYouden Index for each iELISA and ROC analysis was used to determine theROC Curves and AUC. This data is presented in Table 7 below, along withthe 95% confidence intervals for the AUC; Table 7 shows the data fromTable 3 and the additional oligosaccharide data, for ease of comparison.Table 8 shows the probability (P) values for the testing ofstatistically significant differences between the AUC data from eachantigen assay. The AUCs were all compared against each other and the Pvalues was calculated using the Normal Distribution model. Theevaluation was 1-tailed as the prior hypothesis under investigation wasthat antigens that were more ‘1\4’ like would have higher AUC values. Aswith Table 7, Table 8 shows the earlier information from Table 4 withthe additional oligosaccharide data.

A selection of ROC curves are shown in FIG. 12. The Figure shows the ROCcurves for both native antigens tested and amply demonstrates thesuperiority of the B. melitensis 16M OPS over the Y. enterocolitica O:9OPS. The data from the M-like synthetic BSA-pentasaccharide,BSA-tetrasaccharide and BSA-disaccharide conjugate iELISAs is also shownand graphically depicts the improvement in diagnostic performance thatis commensurate with the reduction of α-1,2 linkages in the structuresand retention of the α-1,3 link.

TABLE 7 Performance statistics for OPS and Synthetic OligosaccharideiELISAs as tested on bovine sera Optimal Youden Index % age (YI = DSn +DSp-1) ROC - Area Under Curve α-1,3 YI 95% Confidence Assay linksEstimate DSn % DSp % AUC Interval Y. enterocolitica 0 0.1297 64.44 48.530.5065 0.3964-0.6167 O:9 OPS iELISA B. melitensis 16M 20 0.4232 55.5686.76 0.7065 0.6024-0.8106 OPS iELISA Nonasaccharide 12.5 0.5713 68.8988.24 0.8317 0.7512-0.9122 iELISA Pentasaccharide 25 0.6174 95.56 66.180.8667 0.8019-0.9314 iELISA Trisaccharide 50 0.6091 73.33 86.76 0.86720.7976-0.9367 (terminal α-1,2) iELISA oxiTSM iELISA 33.3/50 0.6670 73.3392.65 0.8948 0.8345-0.9550 Trisaccharide 50 0.7268 80.00 92.65 0.89870.8334-0.9640 (terminal α-1,3) iELISA Tetrasaccharide 33.3 0.6745 73.3394.12 0.9046 0.8478-0.9613 iELISA Disaccharide 100 0.7860 84.44 92.650.9310 0.8345-0.9550 iELISA

The data presented in Table 7 show the antigens in ascending AUC value(lowest at the top and highest at the bottom). In this context, the AUCrepresents the ability of the assay to correctly classify samples fromanimals that are Brucella infected and those that are not. In this dataanalysis, all the samples from animals that were not Brucella infectedwere falsely positive in one or more conventional serodiagnostic assaysfor brucellosis. The results (based on the AUC data) show that the B.melitensis OPS is superior to the Y. enterocolitica O:9 OPS and it isproposed that this is due to the presence of α-1,3 links within the OPSof the former. The percentage of links within the antigenic structuresthat are α-1,3 (the remainder being α-1,2) is also shown in the table.

In general, the AUC values increase with the increase in the percentageof α-1,3 links within the antigen (and decrease in α-1,2 links). This isclearly evident in the comparison of the two native OPS antigens and isalso evident in the AUC data for the nonasaccharide, pentasaccharide,tetrasaccharide and disaccharide BSA conjugates. There are, however,some nuances within the data that should be considered as describedbelow with regard to the comparison between the nonasaccharide BSAconjugate and B. melitensis 16M OPS, the oxidised tetrasaccharideantigen and the two trisaccharide BSA conjugates.

All the synthetic BSA conjugated oligosaccharide antigens have superiordiagnostic capability in this regard compared to the native OPSantigens. This includes the nonasaccharide BSA-conjugate which hasproportionally fewer α-1,3 links than the B. melitensis 16M OPS. Thisapparent anomaly may due to do with the precise positioning of the α-1,3links within the native structure and the multivalent nature in whichantibodies may bind this structure.

It is not straightforward to evaluate the performance of the oxiTSMantigen relative to the others investigated due to the methodologicaldifferences, not least the breaking of the terminal perosamine, linkingdirectly to a functionalised ELISA plate surface from the remnants ofthis structure and thus presenting the reducing sugar as the tip of theantigen. For this reason, the percentage of α-1,3 links presented inTable 7 is shown as 33.3/50 dependent upon the undetermined significanceof the link to the oxidised terminal “perosamine”. Despite theseapparent impediments, the oxiTSM iELISA possessed a greater AUC valuethan the BSA-pentasaccharide and α-1,2 terminated BSA-trisaccharideconjugate iELISAs. This may be due to the loss of the terminal end ‘tip’epitope, since this is not presented by the oxiTSM antigen due to theoxidation and conjugation of the terminal sugar. The natural ‘tip’antigen would be similar in the OPS from A and M dominant Brucella andY. enterocolitica O:9. This ‘tip’ epitope may also explain the higherAUC value for the α-1,3 terminated trisaccharide compared to the α-1,2terminated trisaccharide. According to the structural scheme recentlypresented (Kubler-Kielb & Vinogradov (2013) Carbohydr. Res. 378,144-147), the tip of most OPS molecules in M dominant OPS is a α-1,2linked disaccharide, as would also be the case in the OPS from Adominant Brucella strains and from Y. enterocolitica O:9. The α-1,3terminated trisaccharide does not present such a tip and thus commonanti-‘tip’ epitope antibodies may be less likely to bind in comparisonto the antibodies against the linear M epitope.

The highest AUC value is generated by the BSA-disaccharide conjugateiELISA. This was significantly higher (P=0.0322) than the AUC value forthe BSA-pentasaccharide conjugate iELISA which was itself significantlyhigher than the AUC value derived from the native antigens (Y.enterocolitica O:9 and B. melitensis 16M OPS) The disaccharide has noα-1,2 links present, just a single α-1,3 link. On the basis of thisdata, this structure is highly functional and represents the minimalsize M epitope. The ability of such a small structure to bind to so manypolyclonal antibodies and to do so in such a selective manner is asurprising finding. The negative impact of even a single α-1,2 link wasjust as unexpected. The ability and the extent to which the disaccharide(and the other M-like oligosaccharides), can selectively bind topolyclonal sera raised by infection with A dominant strains of Brucellawas also unexpected, but has been demonstrated here for the first time.

The data from the Max YI values largely agree with the AUC data in thatthe smaller BSA-oligosaccharides conjugates with fewer α-1,2 linksprovide superior diagnostic parameters. The main difference is thesuperior Max YI value for the α-1,3 terminated BSA-trisaccharideconjugate compared to the BSA-tetrasaccharide conjugate. This can berationalised by the arguments put forward above with regards to thereduction of the ‘tip’ epitope as well as the elimination of a α-1,2link.

All of the BSA-oligosaccharide conjugate iELISAs were also applied tothe sera from the eight animals, described above, that wereexperimentally infected with either B. abortus strain 544 (n=4) or Y.enterocolitica O:9 (n=4). Only samples collected from weeks 3, 7, 16 and24 were tested because the samples collected from the B. abortusinfected animals at week 53 gave ambivalent results with conventionalserodiagnostic assay.

The results for the samples from the experimentally infected cattle areshown in FIGS. 13 to 19. FIG. 13 shows the results from B. melitensis16M OPS iELISA and demonstrates that there is a considerable responsefrom the sera derived from the Y. enterocolitica O:9 infected animals.FIGS. 14 and 15 show the data derived from the nonasaccharide andpentasaccharide BSA conjugate iELISAs respectively. The results from theB. melitensis 16M OPS iELISA and pentasaccharide BSA conjugate iELISAare shown against each other in a simple scatter plot in FIG. 16. FIGS.17 and 18 show the data derived from the tetrasaccharide anddisaccharide BSA conjugate iELISAs respectively. Although the ability todifferentiate between the antibodies derived from the two infectiontypes is not absolute, FIG. 18 shows that the disaccharide is close toachieving this aim. This differentiation is more readily visible in thescatter plot, FIG. 19, showing the results from the tetrasaccharide anddisaccharide BSA conjugate iELISAs.

All the serological data from the samples from the experimentallyinfected animals (weeks 3, 7, 16 and 24) were evaluated by the followingquantitative criteria. The percentage (of 16) samples from the cattleexperimentally infected with Y. enterocolitica O:9 with quantitativelygreater serological titres than the lowest titre sample from cattleexperimentally infected with B. abortus strain 544 was calculated. Thepercentage is shown, for each serodiagnostic assay, in Table 9. The CFT,SAT, sLPS iELISA and cELISA and FPA data has been published previously(McGiven et al. (2008) J. Immun. Meth. 20, 7-15) and shows thesignificant degree of cross reaction that occurs with conventional andcontemporary serology.

Although the data set is relatively small, the results show that thedisaccharide and the trisaccharide BSA conjugate iELISAs (using thetrisaccharide with a α-1,3 link at the non-conjugated terminus) were thebest at differentiating between antibodies derived from the two types ofinfection. This demonstrates that as well as providing improveddiagnostic specificity when testing field FPSRs, the disaccharide hasthe same beneficial effect with the samples that have beenexperimentally infected with a Gram-negative bacteria in possession ofthe OPS structure that is most similar to that of Brucella. In thissample set, the results from the nonasaccharide, pentasaccharide,tetrasaccharide and α-1,2 terminated trisaccharide BSA conjugate iELISAsshowed no advantage over the B. melitensis 16M OPS iELISA. The two worstperforming assays in this respect, both with a percentage of 81.25, werethe iELISAs performed using the sLPS from B. abortus S99 (an A dominantstrain) and the Y. enterocolitica O:9 OPS iELISA. This reflects the highdegree of similarity between the OPS structures of these organisms.

TABLE 9 Percentage (of 16) samples from cattle experimentally infectedwith Y. enterocolitica O:9 with quantitatively greater serologicaltitres than the lowest titre sample from cattle experimentally infectedwith B. abortus strain 544 Percentage of samples Classical tests andnative antigens Complement Fixation Test 31.25 Serum Agglutination Tests56.25 iELISA (sLPS, B. abortus S99) 81.25 cELISA (sLPS, B. melitensis16M) 43.75 Fluorescence Polarisation Assay 50.00 Y. enterocolitica O:9OPS iELISA 81.25 B. melitensis 16M OPS iELISA 56.25 Syntheticoligosaccharide antigens Nonasaccharide 56.25 Pentasaccharide 75.00Tetrasaccharide 75.00 Trisaccharide (terminal 1,2) 68.75 Trisaccharide(terminal 1,3) 18.75 Disaccharide 12.50

The nonasaccharide, pentasaccharide, tetrasaccharide and disaccharideBSA-conjugate iELISAs were also evaluated against 125 serum samples from125 randomly sampled non-Brucella infected cattle. The results arepresented by scatter plot, with the results from the Brucella infectedcattle (n=45), in FIGS. 20 (nonasaccharide and pentasaccharide) and 21(tetrasaccharide and disaccharide).

TABLE 10 Performance statistics for Synthetic Oligosaccharide iELISAs astested on bovine sera (randomly sampled non-Brucella infected) OptimalYouden Index ROC - Area Under Curve (YI = DSn + DSp-1) 95% YI DSn DSpConfidence Assay Estimate % % AUC Interval Nonasaccharide 0.9920 100.099.20 0.9998 0.9992-1.000  iELISA Penta- 1.0000 100.0 100.0 1.00001.000-1.000 saccharide iELISA Tetrasaccharide 0.9476 95.56 99.20 0.99640.9911-1.000  iELISA Disaccharide 0.8142 82.22 99.20 0.91080.8401-0.9814 iELISA

The results, shown in scatter plot FIGS. 20 and 21 and Table 10,demonstrate that the nonasaccharide, pentasaccharide and tetrasaccharideBSA-conjugate iELISAs are highly effective serodiagnostic assays. Thedata also suggests that the disaccharide BSA-conjugate iELISA is lesseffective in differentiating between these sample types than the other,larger, oligosaccharides in possession of α-1,2 links. When thedisaccharide BSA-conjugate is used, the sera from the randomly samplednon-Brucella infected cattle have a much more similar response to thatobserved for the FPSR samples than is the case with the nonasaccharide,pentasaccharide and tetrasaccharide BSA-conjugate iELISAs.

The oxiTSM, BSA-pentasaccharide and BSA-nonasaccharide antigens havealso been applied to the detection of specific anti-Brucella antibodiesin small ruminant sera. The oxiTSM antigen was conjugated to theCarbo-BIND™ ELISA plates by the same method as described above and theBSA conjugated oligosaccharides were coated to ELISA plates by the samemethod as described above. The assay was completed using the same methodas described for bovine sera above except for the use of a protein G HRPconjugate. In total, 61 samples were evaluated from individual sheep andgoats from flocks confirmed as infected with B. melitensis biovar 3(mixed ‘A’ and ‘M’ dominance) and positive in iELISA using B. melitensissLPS antigen. Also tested were 94 sera from sheep and goats from GreatBritain, that has always been free of B. melitensis (FIG. 22).

In FIG. 22, the lowest x-axis value for a sample from the Brucellainfected population is 32.5, so there is no data from this populationhidden in the overplotting of the data from the non-Brucella infectedpopulation. The optimised YI value for the 16M OPS iELISA was 0.984 (95%CI=0.952−1.000) and that of the ‘M’ tetrasaccharide iELISA was 0.816(95% CI=0.720−0.912). The 95% confidence intervals for two YI values donot overlap, demonstrating a significant difference in diagnosticperformance. However, the data does demonstrate that the TSM antigendoes detect anti-Brucella antibodies within sera from small ruminantsinfected with B. melitensis. The data in FIG. 23 demonstrates theeffectiveness of the universal nonasaccharide conjugate antigen as,based on this sample set, the DSn and DSp were both 100%.

Finally, ELISA plates were coated with co-povidone disaccharideconjugates 99a and 99b as described above for BSA conjugates. Serial √10dilutions of human sera from a patient infected with Brucella suis wasapplied to the plate and bound antibody was detected by a goat antihuman horse radish peroxidase conjugate. The results are shown in FIG.24. This demonstrated that the disaccharide can detect infection byBrucella in a sample from a human patient.

Antibody Binding Studies

BSA conjugates 98 (comprising the oligosaccharide of Formula XIV) andcopovidone conjugate 100b (comprising an oligosaccharide which isexclusively α-1,2 linked) were each coated on ELISA plates as describedabove. Serial √10 dilutions of mouse monoclonal antibodies that are A orM specific were applied to the plate and bound antibody was detected bya goat anti mouse IgG horse radish peroxidase conjugate. AntibodiesYsT9.1 and YsT9.2 are anti-A antibodies. Antibodies BM10 and BM28 areM-specific. The results are shown in FIGS. 25 and 26 and demonstrate thebinding specificity of the disaccharide, as well as the failure of anexclusively α-1,2 linked oligosaccharide conjugate to bind to an anti-Mantibody.

To evaluate the binding of an anti-M specific mAb to theBSA-tetrasaccharide, trisaccharide and disaccharide conjugates, theywere each coated to ELISA plates at 5 μg per ml (in pH 10.0 carbonatebuffer), double diluted in neighbouring ELISA plate wells and the plateswere incubated and washed as described previously for the bovine serumiELISAs with the BSA conjugates. A working strength dilution ofHRP-conjugated BM40 mAb was added to each well and this was incubatedfor 30 mins at room temperature and then washed as described previously.The plates were developed with HRP ABTS substrate and the reactionstopped after 15 mins by addition of sodium azide. The ELISA plates wereread at 405 nm to measure the optical density of each well.

The results (FIG. 27) show that the BM40 mAb binds to the BSAconjugates; most effectively to the trisaccharide and also, almostequally, to the tetrasaccharide and disaccharide BSA conjugates. Thebinding of the BM40 mAb to these three BSA-oligosaccharide structuresdemonstrates the presence of the M epitope within them as BM40 has beenpreviously shown to be highly specific to M dominant OPS. As well asbinding BM40 mAb, the inventors have shown that these structures alsobind polyclonal sera. Therefore, it would be possible to simultaneouslyincubate both the HRP conjugated BM40 mAb and serum antibodies in orderto create a competitive ELISA for the detection of polyclonal antibodiesin serum. The ability to do this with the disaccharide is particularlynoteworthy as this is already the antigen that provides the bestresolution of FPSRs, incorporating a competitive element into theimmunoassay is expected to yield further improvements in diagnosticcapability.

Vaccination Study

A vaccine glycoconjugate comprising an oligosaccharide formed byexclusively -(1-2)- linked 4,6-dideoxy-4-acylamido-α-pyranose units, theoligosaccharide covalently linked to a carrier protein such as tetanustoxin, is evaluated for vaccine efficacy as follows. The procedures arebased upon the standard model of vaccine efficacy testing as describedwith the OIE Manual of Diagnostic Tests and Vaccines (Nielsen et al.(2009) In: Manual of Diagnostic Tests & Vaccines for Terrestrial Animals2009; Office International Des Epizooties, Paris, Chapter: Bovinebrucellosis, pg 22-29). Control and challenge groups of mice areestablished for evaluation. For example, control group 1—‘vaccination’with PBS only; control group 2—vaccination with unconjugated ‘A’oligosaccharide and tetanus toxoid plus adjuvant; control group3—subcutaneous vaccination with 1×10⁵ CFU per mouse of reference vaccineB. abortus strain S19. The vaccine challenge groups are group4—vaccination with type ‘A’ oligosaccharide tetanus toxoidglycoconjugate; and group 5—vaccination with type ‘M’ oligosaccharidetetanus toxoid glycoconjugate. Following vaccination mice are challengedby intraperitoneal inoculation with 2×10⁵ CFU (per mouse) of B. abortusstrain 544. At 15 days post challenge mice are euthanised and the numberof Brucella cells within their spleens enumerated to provide a metric ofprotection. The mice are also bled and serology performed to determinethe antibody response from each group to the ‘A’ and ‘M’ epitopes. Thisprovides a means by which to evaluate the DIVA potential of theoligosaccharide tetanus toxoid glycoconjugate.

1. (canceled)
 2. A diagnostic conjugate comprising an oligosaccharidewhich comprises at least two units of 4,6-dideoxy-4-acylamido-α-pyranoseand comprising at least one -(1-3)- link between adjacent4,6-dideoxy-4-acylamido-α-pyranose units, in which the carbon atposition 5 in the pyranose is linked to an R group, where R isindependently selected from —CH2OH, —H or an alkyl group having at leastone C atom, the oligosaccharide being covalently linked to anon-saccharide molecule or to a solid entity.
 3. The diagnosticconjugate according to claim 2 wherein the oligosaccharide comprises nomore than one -(1-3)- link.
 4. The diagnostic conjugate according toclaim 2, wherein the oligosaccharide consists of two, three or four4,6-dideoxy-4-acylamido-α-pyranose units.
 5. The diagnostic conjugateaccording to claim 4 which is a specific M-antigen, capable ofpreferentially binding to an anti-M antibody. 6.-8. (canceled)
 9. Thediagnostic conjugate according to claim 2 wherein the oligosaccharidecomprises at least six 4,6-dideoxy-4-acylamido-α-pyranose units.
 10. Thediagnostic conjugate according to claim 9 which is a universal antigen,capable of binding to an anti-M antibody or to an anti-A antibody or toan anti-C/Y antibody.
 11. The diagnostic conjugate according to claim 2wherein the oligosaccharide is selected from the group consisting of adisaccharide of Formula I, a trisaccharide of Formula III or IV, atetrasaccharide of Formula VII, a hexasaccharide of Formula XIII or XIV,and a nonasaccharide of Formula XV; wherein Formula I is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranose;Formula III is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose;Formula IV is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranose;Formula VII is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose;Formula XIII is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose;Formula XIV is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose;and Formula XV is4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose.12. (canceled)
 13. The diagnostic conjugate according to claim 2comprising an oligosaccharide which comprises at least six4,6-dideoxy-4-acylamido-α-pyranose units and comprising overlappingtetrasaccharides of Formula VII, such that a third and fourth4,6-dideoxy-4-acylamido-α-pyranose units in one tetrasaccharide form afirst and second 4,6-dideoxy-4-acylamido-α-pyranose units in the nexttetrasaccharide, to form an oligosaccharide in which the links betweencontiguous 4,6-dideoxy-4-acylamido-α-pyranose units are alternating-(1,2)- and -(1,3)- links; in which the carbon at position 5 in thepyranose is linked to an R group, where R is independently selected from—CH2OH, —H or an alkyl group having at least one C atom.
 14. Thediagnostic conjugate according to claim 2 comprising an oligosaccharidewhich comprises at least seven 4,6-dideoxy-4-acylamido-α-pyranose unitsand comprising overlapping tetrasaccharides of Formula VII, such that afourth 4,6-dideoxy-4-acylamido-α-pyranose unit in one tetrasaccharideforms the a 4,6-dideoxy-4-acylamido-α-pyranose unit in the nexttetrasaccharide; in which the carbon at position 5 in the pyranose islinked to an R group, where R is independently selected from —CH2OH, —Hor an alkyl group having at least one C atom.
 15. The diagnosticconjugate according to claim 2 wherein, in the oligosaccharide, R ismethyl, ethyl or butyl.
 16. (canceled)
 17. The diagnostic conjugateaccording to claim 2 wherein at least one4,6-dideoxy-4-acylamido-α-pyranosyl unit is4,6-dideoxy-4-formamido-α-D-mannopyranosyl.
 18. The diagnostic conjugateaccording to claim 2, wherein the non-saccharide molecule is selectedfrom the group consisting of a protein, Bovine Serum Albumin (BSA), anon-protein carrier molecule comprising hydrophobic elements, and afluorophore.
 19. The diagnostic conjugate according to claim 2, whereinthe oligosaccharide is linked to a solid entity via a protein, viaBovine Serum Albumin (BSA), or via hydrazone conjugation. 20.-35.(canceled)
 36. A kit comprising a diagnostic conjugate according toclaim 2 and further comprising means for obtaining and/or containing abiological sample from an animal, and/or one or more laboratory reagentsuseful for conducting an antibody-antigen binding detection assay,and/or packaging materials and/or materials providing instructions foruse of the kit. 37.-43. (canceled)